Antimicrobial peptides and derived metapeptides

ABSTRACT

The peptides and derivative metapeptides based upon natural antimicrobial peptides have potent and broad spectrum activity against pathogens exhibiting multiple antibiotic resistance. Specific peptides can also potentiate the antimicrobial functions of leukocytes, such as neutrophils. In addition, they exhibit lower inherent mammalian cell toxicities than conventional antimicrobial peptides, and overcome problems of toxicity, immunogenicity, and shortness of duration of effectiveness due to biodegradation, retaining activity in plasma and serum. The peptides and derivative metapeptides exhibit rapid microbicidal activities in vitro, can be used to potentiate conventional antimicrobial agents, to potentiate other antimicrobial peptides and are active against many organisms that exhibit resistance to multiple antibiotics currently in existence.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/911,693, filed Oct. 25, 2010, which is a continuation of U.S. patentapplication Ser. No. 09/648,816, filed Aug. 25, 2000, now U.S. Pat. No.7,820,619, issued Oct. 26, 2010, which is a continuation-in part of U.S.patent application Ser. No. 09/622,561, filed Aug. 18, 2000, nowabandoned, which is a 371 conversion of PCT Application No.PCT/US99/03350, filed Feb. 17, 1999, which application claims priorityto Ser. No. 09/025,319, filed Feb. 18, 1998, now abandoned, all of whichapplications are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to antimicrobial agents, and morespecifically pertains to peptides useful as antimicrobial agents for theprevention and treatment of infections caused by organisms, such asbacteria and fungi, many of which are resistant to conventionalantibiotics.

2. Description of Related Art

Human infections due to antibiotic-resistant bacteria and fungi areincreasing in frequency and severity. Microbial pathogens exhibitingresistance to one or more antibiotics can now commonly be found incommunity and nosocomial settings. Antibiotic resistant pathogenscurrently of the greatest concern are methicillin (multiple) resistantStaphylococcus aureus (MRSA), vancomycin intermediate-resistant S.aureus (VISA) or vancomycin-resistant S. aureus (VRSA), vancomycinresistant Enterococcus faecalis or Enterococcus faecium (VRE), multidrug-resistant Streptococcus pneumoniae (MDRSPn) or Streptococcuspyogenes (MDRSPy), multidrug-resistant Pseudomonas aeruginosa (MDRPA),and azole resistant Candida albicans (ARCA).

Antimicrobial peptides have heretofore generally been considered to haveundesirable toxicity, immunogenicity, and short half-lives due tobiodegradation. However, endogenous antimicrobial peptides are believedto be integral to non-oxidative mechanisms of antimicrobial hostdefense. Stable, peptide-resistant mutants are rare, likely becausemicrobicidal peptides appear to target the cytoplasmic membrane or otheressential structures and/or functions of pathogens.

Investigations conducted over the past decade have demonstrated theexistence of potent microbicidal peptides from various mammaliantissues. Perhaps the most thoroughly studied among these are defensinsfrom neutrophil azurophilic granules. Related peptides such asβ-defensins and cryptdins have also been isolated and characterized. Todate, nearly 20 distinct defensins have been found in mammalianneutrophils.

Aside from neutrophils, the probability that platelets play an integralrole in host defense against infection has been demonstrated by thefollowing facts: i) platelets are the earliest and predominant cells atsites of microbial infection of vascular endothelium; ii) plateletsadhere to and internalize microbial pathogens; iii) bacterial, fungal,and protozoal pathogens are damaged or killed by activated platelets invitro; iv) thrombocytopenia increases susceptibility to and severity ofsome infections; v) rabbit and human platelets release plateletmicrobicidal proteins (PMPs) when stimulated with microorganisms orplatelet agonists integral to infection in vitro; and vi) PMPs exertrapid and potent microbicidal activities against a broad spectrum ofpathogens in vitro. It has been hypothesized that PMPs substantiallycontribute to platelet antimicrobial host defense by direct microbicidalactions, and may amplify cell mediated immune mechanisms such asneutrophil microbicidal activity. Similar to defensins, PMPs appear todisrupt microbial cytoplasmic membranes to achieve microbicidalactivity. Present data indicates that PMP-2 (Sequence No. 1), tPMP-1,and defensin hNP-1 employ distinct mechanisms, and that thesedifferences are related to differences in protein structure.

The majority of known mammalian antimicrobial peptides have beenlocalized within leukocytes (e.g., defensins), or secreted ontoepithelial surfaces such as intestinal lumen or tracheal epithelium(e.g., cryptdins, tracheal antimicrobial peptide). Prohibitive levels ofmammalian cell toxicity have been noted with many of these peptides whenthey have been tested as antimicrobial therapeutics. In contrast, PMPsexert potent in vitro microbicidal activity against a broad spectrum ofbacteria and fungi under physiological conditions that exist in theintravascular space. Several PMPs are released from platelets stimulatedwith agonists associated with infection. Therefore, in response totissue injury, PMPs are likely released into the mammalian bloodstreamat localized sites of infection. In preliminary studies, tPMP-1 andPMP-2 (Sequence No. 1) have been found to cause minimal damage of humanerythrocytes or vascular endothelial cells in vitro as compared withdefensin hNP-1. In addition, PMPs exert potent microbicidal activityagainst bacterial and fungal pathogens, comparable to defensins whichhave been observed at concentrations as low as 0.5 μg/ml in vitro. Thesepotencies compare favorably to potent conventional antimicrobial agentssuch as aminoglycosides or amphotericin B.

A large family of antimicrobial peptides from mammalian platelets hasalso been isolated, and amino acid compositions and primary structuresof endogenous antimicrobial peptides originating from mammalian andnon-mammalian tissues now constitute a database of over 300antimicrobial peptides. Recent advances in peptide structural analyseshave provided important new information regarding the relationshipbetween structure and microbicidal activities among these peptides. Forexample, the fact that many antimicrobial peptides are small, cationic,and contain amphiphilic α-helical domains is well established.

It would be desirable to provide peptides that are active againstorganisms that exhibit resistance to antibiotics, for use eitherindependently or in combination to potentiate conventional antimicrobialagents or other antimicrobial peptides and/or which potentiate theantimicrobial functions of leukocytes. It is also desirable to providemicrobicidal peptides that are based upon natural antimicrobialpeptides, to overcome problems of toxicity and immunogenicity. Toovercome short half-life due to degradation, such peptides should beresistant to proteolytic degradation, and should be stable intemperatures as high as 80° C., and in extremes of alkalinity andacidity, ranging from about pH 2 to about pH 10, for example. It isfurther desirable that such peptides should be amenable to chemicalsynthesis or recombinant DNA-based expression, facilitating theirproduction in quantities necessary for testing or therapeuticapplication. The present invention addresses, at least in part, theseand other needs.

SUMMARY OF THE INVENTION

Briefly, and in general terms, the present invention provides forpeptides and derivative metapeptides (peptides derived from primarypeptide templates) that likely target the microbial cytoplasmicmembrane, leading to perturbation of the membrane. These effects almostcertainly lead to ensuing effects on intracellular targets. This, alongwith secondary effects on intracellular functions such as macromolecularsynthesis or bioenergetics, leads to overall cellular disruption andrapid death of the targeted microbes.

The invention accordingly provides for antimicrobial peptides forpotentiating antimicrobial agents active against pathogenic organismssuch as bacteria and fungi. The present invention provides for 1) novelantimicrobial peptides that act directly on the pathogen to exertmicrobicidal or microbiostatic activity; 2) novel antimicrobial peptidesthat potentiate one or more antimicrobial activities of leukocytes; and3) novel antimicrobial peptide mosaics that combine such direct andleukocyte potentiating activities. In one presently preferredembodiment, the antimicrobial peptide comprises a peptide having anamino acid sequence selected from the group of amino acid sequencesconsisting essentially of a first peptide template XZBZBXBXB andderivatives thereof selected from the group consisting of XZBBZBXBXB,BXZXB, BXZXZXB, XBBXZXBBX, and BBXZBBXZ, and a second peptide templateXBBXX and derivatives thereof selected from the group consisting ofXBBXBBX, XBBXXBBX, BXXBXXB, XBBZXX, XBBZXXBB, and XBBZXXBBXXZBBX, whereB is at least one positively charged ammo acid, X is at least onenon-polar, hydrophobic amino acid, and Z is at least one aromatic aminoacid. In a presently preferred aspect of the invention, B is selectedfrom the group of amino acids consisting of lysine, arginine, histidine,and combinations thereof; X is selected from the group of amino acidsconsisting of leucine, isoleucine, alanine, valine, and combinationsthereof; and Z is selected from the group of amino acids consisting ofphenylalanine, tryptophan, tyrosine and combinations thereof. Otheramino acids, including glutamine, asparagine, proline, cystine, asparticacid, glutamic acid, glycine, methionine, serine and threonine, may beinterplaced within these primary structural motifs in a given case. Inanother aspect, the peptide or derived metapeptide of the invention canfurther comprise D-isomeric amino acids. In another aspect, the peptideor derived metapeptide of the invention can further comprise aretromeric sequence of amino acids. In a further aspect, the peptide orderived metapeptide of the invention can further comprise a modifiedamino acid group selected from the group consisting ofN-^(∈)monomethyl-lysine, (β-branched, N-methyl, α,β-dehydro,α,α-dialkyl, fluorinated amino acids, and combinations thereof in director retromeric sequences. The antimicrobial peptides can also betruncations, extensions, combinations or fusions of the templatepeptides disclosed. Despite these variations, the disclosed peptideswill adhere to the general structural motifs indicated, therebypreserving their uniqueness. In a preferred embodiment, the total lengthof the peptides of the invention will be less than about 150 residues,and the total length preferably will be approximately 5 to 150 residues.

In another aspect of the invention, antimicrobial peptides and derivedmetapeptides that potentiate antimicrobial activity of leukocytes andare active alone or in combination with other agents directly againstorganisms such as bacteria and fungi can comprise peptides having aminoacid sequences selected from the group consisting essentially ofcombined amino acid sequences AL and LA, wherein A represents anantimicrobial domain consisting essentially of a first peptide templateXZBZBXBXB and derivatives thereof selected from the group consisting ofXZBBZBXBXB, BXZXB, BXZXZXB, XBBXZXBBX, and BBXZBBXZ, and a secondpeptide template XBBXX and derivatives thereof selected from the groupconsisting of XBBXBBX, XBBXXBBX, BXXBXXB, XBBZXX, XBBZXXBB, andXBBZXXBBXXZBBX and L represents a leukocyte potentiating domainconsisting essentially of JJJCJCJJJJJJ, and J is selected from X, Z andB. Thus, an example of AL can be: XZBZBXBXBJJJCJCJJJJJJ; and an exampleof LA can be: JJJCJCJJJJJJXZBZBXBXB.

Within one aspect of the present invention antimicrobial peptides areprovided comprising a peptide of from 7 to 74 amino acids containing a 7amino acid core sequence: aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇, wherein aa₁ isthe amino-terminus of the core sequence; one of aa₆ and aa₇ is selectedfrom the group consisting of phenylalanine, tryptophan and tyrosine,such that when aa₆ is phenylalanine aa₇ is selected from the groupconsisting of lysine, arginine and histidine, when aa₆ is tryptophan aa₇is lysine, and when aa₇ is phenylalanine aa₆ is leucine; and retromers,truncations, extensions, combinations, fusions, and derivatives thereof,the peptide having antimicrobial activity. Within one embodiment, aa₁ isselected from the group consisting of alanine, lysine and glycine; aa₂is selected from the group consisting of leucine and arginine; aa₃ istyrosine; and aa₄ and aa₅ are selected from the group consisting oflysine, aa₄ and aa₅ are selected from the group consisting of lysine andarginine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 7 to 74 amino acids containing a 7amino acid core sequence: aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇, wherein aa₁ isthe amino-terminus of the peptide; aa₆ is selected from the groupconsisting of phenylalanine and tryptophan and tyrosine; and aa₇ isselected from the group consisting of lysine and arginine; andretromers, truncations, extensions, combinations, fusions, andderivatives thereof, the peptide having antimicrobial activity. Withinone embodiment of the above aa₆ is selected from the group consisting ofphenylalanine and tryptophan, and aa₇ is selected from the groupconsisting of lysine and arginine. Within other embodiments, aa₁ isselected from the group consisting of alanine, lysine and glycine; aa₂is selected from the group consisting of leucine and arginine; aa₃ istyrosine; and aa₄ and aa₅ are selected from the group consisting oflysine, arginine, glutamine, proline, histidine and asparagine. Withinfurther embodiments aa₄ and aa₅ are selected from the group consistingof lysine and arginine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 13 to 18 amino acids containing a12 amino acid core sequence;aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂, wherein aa₁ is theamino-terminus of the peptide and is selected from the group consistingof leucine, isoleucine, alanine, valine, serine, lysine and glycine; aa₂is selected from the group consisting of leucine, isoleucine, alanine,valine, serine and arginine; aa₃ is selected from the group consistingof phenylalanine, tryptophan and tyrosine; aa₄ and aa₅ are selected fromthe group consisting of lysine, arginine and histidine; one of aa₆ andaa₇ is selected from the group consisting of phenylalanine, tryptophanand tyrosine, such that when aa₆ is phenylalanine aa₇ is selected fromthe group consisting of lysine, arginine and histidine, when aa₆ istryptophan aa₇ is lysine, and when aa₇ is phenylalanine aa₆ is leucine;aa₈ is selected from the group consisting of lysine, arginine, histidineand asparagine; aa₉ is selected from the group consisting of lysine,arginine and histidine; aa₁₀ is selected from the group consisting ofleucine, isoleucine, alanine, valine and serine; aa₁₁ is selected fromthe group consisting of leucine, isoleucine, alanine, valine, serine andlysine; and aa₁₂ is selected from the group consisting of lysine,arginine and histidine; and retromers, truncations, extensions,combinations, fusions, and derivatives thereof, the peptide havingantimicrobial activity. Within a further embodiment aa₁ is selected fromthe group consisting of alanine, lysine and glycine; aa₂ is selectedfrom the group consisting of leucine and arginine; aa₃ is tyrosine; aa₄,and aa₅ are selected from the group consisting of lysine and arginine;aa₈ is selected from the group consisting of lysine and asparagine; aa₉is lysine; aa₁₀ is selected from the group consisting of leucine andisoleucine; aa₁₁ is selected from the group consisting of leucine andlysine; and aa₁₂ is selected from the group consisting of lysine andarginine. Within yet further embodiments one of aa₆ and aa₇ is selectedfrom the group consisting of phenylalanine and tryptophan, such thatwhen aa₆ is phenylalanine aa₇ is selected from the group consisting oflysine and arginine, when aa₆ is tryptophan aa₇ is lysine, and when aa₇is phenylalanine aa₆ is leucine. Within other embodiments aa₁ isselected from the group consisting of alanine, lysine and glycine; aa₂is selected from the group consisting of leucine and arginine; aa₃ istyrosine; aa₆ is selected from the group consisting of phenylalanine,tryptophan and tyrosine; aa₇ is selected from the group consisting oflysine and arginine; aa₈ is selected from the group consisting of lysineand asparagine; aa₉ is lysine; aa₁₀ is selected from the groupconsisting of leucine and isoleucine; aa₁₁ is selected from the groupconsisting of leucine and lysine; and aa₁₂ is selected from the groupconsisting of aa₆ is phenylalanine aa₇ is lysine or arginine, and whenaa₆ is tryptophan aa₇ is lysine. Within other embodiments aa₆ isselected from the group consisting of phenylalanine, tryptophan andtyrosine; and aa₇ is selected from the group consisting of lysine andarginine. Within yet other embodiments aa₆ is phenylalanine aa₇ isselected from the group consisting of lysine and arginine, and when aa₆is tryptophan aa₇ is lysine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 13 to 18 amino acids containing a13 amino acid core sequence:aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃, wherein aa₁ isthe amino-terminus of the peptide and is selected from the groupconsisting of leucine, isoleucine, alanine, valine, serine, lysine andglycine; aa₂ is selected from the group consisting of leucine,isoleucine, alanine, valine, serine and arginine; aa₃ is selected fromthe group consisting of phenylalanine, tryptophan, tyrosine; aa₄ and aa₅are selected from the group consisting of lysine, arginine andhistidine; one of aa₆ and aa₇ is selected from the group consisting ofphenylalanine, tryptophan, tyrosine, and the other of aa₆ and aa₇ isselected from the group consisting of lysine, arginine and leucine,wherein when aa₆ is phenylalanine aa₇ is selected from the groupconsisting of lysine and arginine, when aa₆ is tryptophan aa₇ is lysine,and when aa₇ is phenylalanine aa₆ is leucine; aa₈ is selected from thegroup consisting of lysine, arginine, histidine and asparagine; aa₉ isselected from the group consisting of lysine, arginine and histidine;aa₁₀ is selected from the group consisting of leucine, isoleucine,alanine, valine and serine; aa₁₁ is selected from the group consistingof leucine, isoleucine, alanine, valine, serine and lysine; and aa₁₂ isselected from the group consisting of lysine, arginine and histidine;and aa₁₃ is selected from the group consisting of leucine, isoleucine,alanine, valine, serine, arginine and phenylalanine; and retromers,truncations, extensions, combinations, fusions, and D-isomeric aminoacid, retromeric, N-monomethyl-lysine, and fluorinated amino acidderivatives thereof, the peptide having antimicrobial activity. Withinone embodiment aa₁ is selected from the group consisting of alanine,lysine and glycine; aa₂ is selected from the group consisting of leucineand arginine; aa₃ is tyrosine; aa₄ and aa₅ are selected from the groupconsisting of lysine, arginine and histidine; aa₈ is selected from thegroup consisting of lysine and asparagine; aa₉ is lysine; aa₁₀ isselected from the group consisting of leucine and isoleucine; aa₁₁ isselected from the group consisting of leucine and lysine; and aa₁₂ isselected from the group consisting of lysine and arginine. Withinanother embodiment aa₁₃ is selected from the group consisting of serine,leucine, arginine and phenylalanine.

Within other aspects of the invention antimicrobial peptides areprovided comprising a peptide of from 13 to 74 containing an amino acidcore sequence selected from the group consisting of truncations of PMP-1(Sequence No. 2), and retromers, extensions, combinations and fusionsthereof; truncations of PMP-2 (Sequence No. 1), and retromers,extensions, combinations and fusions thereof. Within one embodiment theantimicrobial peptide further comprises a pharmaceutically acceptablecarrier. Within other embodiments the peptide is a truncation of PMP-2(Sequence No. 1) and comprises residues 28 to 74 of PMP-2 (Sequence No.1). Within further embodiments the peptide is a truncation of PMP-2(Sequence No. 1) and comprises residues 43 to 74 of PMP-2 (Sequence No.1). Within yet other embodiments the peptide is a truncation of PMP-2(Sequence No. 1) and comprises residues 59 to 74 of PMP-2 (Sequence No.1). Within another embodiment the peptide is a truncation of PMP-2(Sequence No. 1) and comprises residues 45 to 74 of PMP-2 (Sequence No.1). Within a further embodiment the peptide comprises an extension ofRP-1 (Sequence No. 3) by RP-1 residues 1-10. Within an alternativeembodiment the peptide comprises a combination of RP-1 (Sequence No. 3)with RP-13 (Sequence No. 14).

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 8 to 20 amino acids containing anamino acid core sequence of a first amino acid sequence domain, a secondamino acid sequence domain, and a third amino acid sequence domain,where the first amino acid sequence domain is a sequence of from one tosix amino acids selected from the group consisting of leucine,isoleucine, alanine, valine, serine, glycine, and threonine; the secondamino acid sequence domain is a sequence of from one to two amino acidsselected from the group consisting of lysine, arginine, histidine,glutamine, proline, glutamic acid, aspartic acid and glycine; the thirdamino acid sequence domain is a sequence of from one to nine amino acidsselected from the group consisting of leucine, isoleucine, alanine,valine, serine, glycine, and threonine; and where the amino acids withinthe first, second and third amino acid sequence domains may beseparated, and the first, second and third amino acid domains may beseparated from each other by up to three amino acids selected from thegroup consisting of asparagine, cystine, aspartic acid, glutamic acidand methionine; and retromers, truncations, extensions, combinations,fusions, and derivatives thereof, the peptide having antimicrobialactivity. Within one embodiment the peptide contains an amino acid coresequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇,wherein aa₁ is the amino-terminus of the peptide and is selected fromthe group consisting of leucine, isoleucine, alanine, valine, serine,glycine, and threonine; aa₂ is selected from the group consisting ofleucine, isoleucine, alanine, valine, serine, glycine, and threonine;aa₃ and aa₄ are selected from the group consisting of lysine, arginine,histidine, glutamine, and proline; aa₅ is selected from the groupconsisting of asparagine, cystine, aspartic acid, glutamic acid andmethionine; aa₆ is selected from the group consisting of leucine,isoleucine, alanine, valine, serine, glycine, and threonine; aa₇ isselected from the group consisting of lysine, arginine, histidine,glutamine, proline, glutamic acid, aspartic acid and glycine; aa₈ isselected from the group consisting of lysine, arginine, histidine,glutamine, proline and glutamic acid; aa₉, aa₁₁, aa₁₃, aa₁₅, aa₁₆, andaa₁₇ are selected from the group consisting of leucine, isoleucine,alanine, valine, serine, glycine, and threonine; aa₁₀ and aa₁₂ areselected from the group consisting of asparagine, cystine, asparticacid, glutamic acid and methionine; and aa₁₄ is selected from the groupconsisting of lysine, arginine, histidine, glutamine and proline. Withinanother embodiment the peptide contains an amino acid core sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇,wherein aa₁ is the amino-terminus of the peptide core sequence and isalanine; aa₂ is threonine; aa₃ and aa₄ are lysine; aa₅ is asparagine;aa₆ is glycine; aa₇ is arginine; aa₈ is lysine; aa₉, aa₁₁, aa₁₃ and aa₁₇are leucine; aa₁₀ is cystine; aa₁₂ is aspartic acid; aa₁₄ is glutamine,and aa₁₅ and aa₁₆ are alanine. Within other embodiments the peptidecontains an amino acid core sequence aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈,wherein aa₁ is the amino-terminus of the peptide core sequence and isarginine; aa₂ is phenylalanine; aa₃ is glutamic acid; aa₄ is lysine; aa₅is serine; aa₆ is lysine; aa₇ is isoleucine; and aa₈ is lysine. Withinanother embodiment the peptide contains an amino acid core sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀,wherein aa₁ is the amino-terminus of the peptide and is serine; aa₂ isalanine; aa₃ is isoleucine; aa₄ is histidine; aa₅ is proline; aa₆ andaa₇ are serine; aa₈ is isoleucine; aa₉ is leucine; aa₁₀ is lysine; aa₁₁is leucine; aa₁₂ is glutamic acid; aa₁₃ is valine; aa₁₄ is isoleucine;aa₁₅ is cystine; aa₁₆ is isoleucine; aa₁₇ is glycine; aa₁₈ is valine;aa₁₉ is leucine; and aa₂₀ is glutamine. Within further embodiments thepeptide contains an amino acid core sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄, whereinaa₁ is the amino-terminus of the peptide and is tyrosine; aa₂ isalanine; aa₃ is selected from the group consisting of aspartic acid andglutamic acid; aa₄ and aa₅ are selected from the group consisting ofleucine, arginine and histidine; aa₆ is cystine; aa₇ is selected fromthe group consisting of threonine or valine; aa₈ is cystine; aa₉ isserine; aa₁₀ is isoleucine; aa₁₁ is lysine; aa₁₂ is alanine; aa₁₃ isglutamic acid; and aa₁₄ is valine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 5 to 150 amino acids containing anamino acid core sequence of a first amino acid sequence domain, a secondamino acid sequence domain, a third amino acid sequence domain, and afourth amino acid sequence domain, and wherein the first amino acidsequence domain is at the amino-terminus of the amino acid core sequenceand is a sequence of from one to five amino acids selected from thegroup consisting of phenylalanine, tryptophan, tyrosine, where aminoacids of the first amino acid sequence domain may be separated from eachother by an amino acid selected from the group consisting of leucine,isoleucine, alanine, valine and serine; the second amino acid sequencedomain is an amino acid selected from the group consisting of lysine,arginine, histidine, glutamine, and proline; the third amino acidsequence domain is a sequence of from one to five amino acids selectedfrom the group consisting of phenylalanine, tryptophan, tyrosine; andthe fourth amino acid sequence domain is an amino acid selected from thegroup consisting of lysine, arginine, histidine, glutamine, and proline;and retromers, truncations, extensions, combinations, fusions, andderivatives thereof, the peptide having antimicrobial activity. Withinone embodiment the peptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁ is theamino-terminus of the peptide and is lysine; aa₂ is phenylalanine; aa₃is lysine; aa₄ is histidine; aa₅ is tyrosine; aa₆ and aa₇ arephenylalanine; aa₈ is tryptophan; aa₉ is lysine; aa₁₀ is tyrosine; andaa₁₁ is lysine. Within another embodiment the peptide contains the aminoacid sequence aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁is the amino-terminus of the peptide and is lysine; aa₂ is glycine; aa₃is tyrosine; aa₄ is phenylalanine; aa₅ is tyrosine; aa₆ isphenylalanine; aa₇ is leucine; aa₈ is phenylalanine; aa₉ is lysine; aa₁₀is phenylalanine; and aa₁₁ is lysine. Within other embodiments thepeptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁ is theamino-terminus of the peptide and is lysine; aa₂ is tryptophan; aa₃ islysine; aa₄, aa₅, aa₆, aa₇ and aa₈ are tryptophan; aa₉ is lysine; aa₁₀is tryptophan; and aa₁₁ is lysine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 11 to 20 amino acids containingfrom one to four units of an amino acid core sequence domain, whereinadjacent units of the amino acid core sequence domain may be separatedfrom each other by from one to two amino acids selected from the groupconsisting of phenylalanine, tryptophan, tyrosine, asparagine, cystine,aspartic acid, glutamic acid and methionine; wherein the amino acidsequence domain consists of a first group of amino acids and a secondgroup of amino acids, the first group of amino acids consisting of fromone to six amino acids selected from the group of leucine, isoleucine,alanine, valine, serine, glycine, and threonine, and the second group ofamino acids consisting of from one to three amino acids selected fromthe group of lysine, arginine, histidine, glutamine, and proline;wherein the amino acids in the first and second groups of amino acidsmay be separated by from one to two amino acids selected from the groupconsisting of phenylalanine, tryptophan, tyrosine, asparagine, cystine,aspartic acid, glutamic acid and methionine; and wherein the first andsecond groups of amino acids may be separated from each other by anamino acid selected from the group consisting of phenylalanine,tryptophan and tyrosine; and retromers, truncations, extensions,combinations, fusions, and derivatives thereof, the peptide havingantimicrobial activity. Within one embodiment the peptide peptidecomposition of Claim 36, wherein the peptide contains two of the unitsof the amino acid core sequence domain. Within a further embodiment twounits of the amino acid core sequence domain are separated by an aminoacid selected from the group consisting of asparagine, cystine, asparticacid, glutamic acid and methionine, and an amino acid selected from thegroup consisting of phenylalanine, tryptophan and tyrosine. Within afurther embodiment the two units of the amino acid core sequence domainare separated by an amino acid selected from the group consisting ofphenylalanine, tryptophan and tyrosine. Within a related embodiment thepeptide contains three of the units of the amino acid core sequencedomain. Within another embodiment the first and second units of theamino acid core sequence domain are separated by an amino acid selectedfrom the group consisting of phenylalanine, tryptophan and tyrosine.Within a related embodiment, the peptide contains four of the units ofthe amino acid core sequence domain. Within yet other embodiments, thefirst and second units of the amino acid core sequence domain areseparated by an amino acid selected from the group consisting ofphenylalanine, tryptophan and tyrosine.

Within another embodiment the peptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃, wherein aa₁ isthe amino-terminus of the peptide and is proline, aa₂ is arginine, aa₃is isoleucine, aa₄ and aa₅ are lysine, aa₆ is isoleucine, aa₇ is valine,aa₈ is glutamine, aa₉ and aa₁₀ are lysine, aa₁₁ is leucine, aa₁₂ isalanine, and aa₁₃ is glycine. Within a further embodiment, the peptidecontains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉,wherein aa₁ is the amino-terminus of the peptide and is lysine, aa₂ istryptophan, aa₃ is valine, aa₄ is arginine, aa₅ is glutamic acid, aa₆ istryosine, aa₇ is isoleucine, aa₈ is asparagine, aa₉ is serine, aa₁₀ isleucine, aa₁₁ is glutamic acid, aa₁₂ is methionine, aa₁₃ is serine, aa₁₄and aa₁₅ are lysine, aa₁₆ is glycine, aa₁₇ is leucine, aa₁₈ is alanine,and aa₁₉ is glycine. Within a further embodiment the peptide containsthe amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀,wherein aa₁ is the amino-terminus of the peptide and is glutamic acid,aa₂ is tryptophan, aa₃ is valine, aa₄ is glutamine, aa₅ is lysine, aa₆is tryosine, aa₇ is valine, aa₈ is serine, aa₉ is asparagine, aa₁₀ isleucine, aa₁₁ is glutamic acid, aa₁₂ is leucine, aa₁₃ is serine, aa₁₄ isalanine, aa₁₅ is tryptophan, aa₁₆ and aa₁₇ are lysine, aa₁₈ isisoleucine, aa₁₉ is leucine, and aa₂₀ is lysine. Within yet anotherembodiment the peptide contains the amino acid sequence aaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂, wherein aa₁ is theamino-terminus of the peptide and is serine, aa₂ is tryptophan, aa₃ isvaline, aa₄ is glutamine, aa₅ is glutamic acid, aa₆ is tryosine, aa₇ isvaline, aa₈ is tryosine, aa₉ is asparagine, aa₁₀ is leucine, aa₁₁ isglutamic acid, and aa₁₂ is leucine. Within another embodiment thepeptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆,wherein aa₁ is the amino-terminus of the peptide and is alanine, aa₂ isasparagine, aa₃ is serine, aa₄ is glycine, aa₅ is glutamic acid, aa₆ isglycine, aa₇ is asparagine, aa₈ is phenylalanine, aa₉ is leucine, aa₁₀is alanine, aa₁₁ is glutamic acid, aa₁₂, aa₁₃ and aa₁₄ are glycine, aa₁₅is valine, and aa₁₆ is arginine. Within yet another embodiment thepeptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀,wherein aa₁ is the amino-terminus of the peptide and is alanine, aa₂ isasparagine, aa₃ is serine, aa₄ is glycine, aa₅ is glutamic acid, aa₆ isglycine, aa₇ is asparagine, aa₈ is phenylalanine, aa₉ is leucine, aa₁₀is alanine, aa₁₁ is glutamic acid, aa₁₂, aa₁₃ and aa₁₄ are glycine, aa₁₅is valine, aa₁₆ is arginine, aa₁₇ is lysine, aa₁₈ is leucine, aa₁₉ isisoleucine, and aa₂₀ is lysine.

Within further embodiments the peptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃, wherein aa₁ isthe amino-terminus of the peptide and is glutamic acid, aa₂ is glycine,aa₃ is valine, aa₄ is asparagine, aa₅ is aspartic acid, aa₆ isasparagine, aa₇ and aa₈ are glutamic acid, aa₉ is glycine, aa₁₀ and aa₁₁are phenylalanine, aa₁₂ is serine, and aa₁₃ is alanine. Within yetanother embodiment the peptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈,wherein aa₁ is the amino-terminus of the peptide and is lysine, aa₂ isphenylalanine, aa₃ is asparagine, aa₄ is lysine, aa₅ is serine, aa₆ islysine, aa₇ is leucine, aa₈ and aa₉ are lysine, aa₁₀ is threonine, aa₁₁is glutamic acid, aa₁₂ is threonine, aa₁₃ is glutamine, aa₁₄ is glutamicacid, aa₁₅ is lysine, aa₁₆ is asparagine, aa₁₇ is proline, and aa₁₈ isleucine. Within further embodiments the peptide contains the amino acidsequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅,wherein aa₁ is the amino-terminus of the peptide and is alanine, aa₂ isasparagine, aa₃ is leucine, aa₄ is isoleucine, aa₅ is alanine, aa6 isthreonine, aa₇ and aa₈ are lysine, aa₉ is asparagine, aa₁₀ is glycine,aa₁₁ is arginine, aa₁₂ is lysine, aa₁₃ is leucine, aa₁₄ is cystine, andaa₁₅ is leucine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 5 to 150 amino acids having athree amino acid core sequence of a first amino acid which is cystine, asecond amino acid which is selected from the group consisting ofleucine, isoleucine, alanine, valine, serine, glycine, threonine,phenylalanine, tryptophan, tyrosine, lysine, arginine, glutamine,proline, and histidine, and a third amino acid which is cystine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 5 to 150 amino acids having anamino acid core sequence of a first amino acid sequence domain, a secondamino acid sequence domain, and a third amino acid sequence domain,wherein the first amino acid sequence domain is a sequence of threeamino acids selected from the group consisting of leucine, isoleucine,alanine, valine, serine, glycine, threonine, phenylalanine, tryptophan,tyrosine, lysine, arginine, glutamine, proline, and histidine; thesecond amino acid sequence is a first amino acid which is cystine, asecond amino acid which is selected from the group consisting ofleucine, isoleucine, alanine, valine, serine, glycine, threonine,phenylalanine, tryptophan, tyrosine, lysine, arginine, glutamine,proline, and histidine, and a third amino acid which is cystine; and thethird amino acid sequence is a sequence of six amino acids selected fromthe group consisting of leucine, isoleucine, alanine, valine, serine,glycine, threonine, phenylalanine, tryptophan, tyrosine, lysine,arginine, glutamine, proline, and histidine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 5 to 150 amino acids having anamino acid core sequence of a first amino acid sequence domain, a secondamino acid sequence domain, a third amino acid sequence domain, and afourth amino acid sequence domain, wherein the first amino acid sequencedomain is a sequence of from 13 to 18 amino acids containing a 12 aminoacid core sequence: aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂,wherein aa₁ is the amino-terminus of the peptide, one of aa₆ and aa₇ isselected from the group consisting of phenylalanine and tryptophan, suchthat when aa₆ is phenylalanine aa₇ is selected from the group consistingof lysine and arginine, when aa₆ is tryptophan aa₇ is lysine, and whenaa₇ is phenylalanine aa₆ is leucine; the second amino acid sequencedomain is a sequence of three amino acids selected from the groupconsisting of leucine, isoleucine, alanine, valine, serine, glycine,threonine, phenylalanine, tryptophan, tyrosine, lysine, arginine,glutamine, proline, and histidine; the third amino acid sequence domainis a first amino acid which is cystine, a second amino acid which isselected from the group consisting of leucine, isoleucine, alanine,valine, serine, glycine, threonine, phenylalanine, tryptophan, tyrosine,lysine, arginine, glutamine, proline, and histidine, and a third ammoacid which is cystine; and the fourth amino acid sequence domain is asequence of six amino acids selected from the group consisting ofleucine, isoleucine, alanine, valine, serine, glycine, threonine,phenylalanine, tryptophan, tyrosine, lysine, arginine, glutamine,proline, and histidine.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 5 to 150 amino acids having anamino acid core sequence of a first amino acid sequence domain, a secondamino acid sequence domain, a third amino acid sequence domain, and afourth amino acid sequence domain, wherein the first amino acid sequencedomain is a sequence of three amino acids selected from the groupconsisting of leucine, isoleucine, alanine, valine, serine, glycine,threonine, phenylalanine, tryptophan, tyrosine, lysine, arginine,glutamine, proline, and histidine; the second amino acid sequence is afirst amino acid which is cystine, a second amino acid which is selectedfrom the group consisting of leucine, isoleucine, alanine, valine,serine, glycine, threonine, phenylalanine, tryptophan, tyrosine, lysine,arginine, glutamine, proline, and histidine, and a third amino acidwhich is cystine; the third amino acid sequence is a sequence of sixamino acids selected from the group consisting of leucine, isoleucine,alanine, valine, serine, glycine, threonine, phenylalanine, tryptophan,tyrosine, lysine, arginine, glutamine, proline, and histidine; and thefourth amino acid sequence domain is a sequence of from 13 to 18 aminoacids containing a 12 amino acid core sequence:aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂, wherein a₁, is theamino-terminus of the peptide, one of aa₆ and aa₇ is selected from thegroup consisting of phenylalanine and tryptophan, such that when aa₆ isphenylalanine aa₇ is selected from the group consisting of lysine andarginine, when aa₆ is tryptophan aa₇ is lysine, and when aa₇ isphenylalanine aa₆ is leucine, and retromers, truncations, extensions,combinations, fusions, and derivatives thereof, the peptide havingantimicrobial activity.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 11 to 22 amino acids containing an10 amino acid core sequence: aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀,wherein aa₁ is the amino-terminus of the amino acid core sequence and isthreonine; aa₂ and aa₃ are selected from the group consisting of lysineand arginine; aa₁ is asparagine; aa₅ is glycine; aa₆ is selected fromthe group consisting of lysine, arginine, glutamic acid and glycine; aa₇is selected from the group consisting of lysine, arginine and glutamicacid; aa₈ is leucine; aa₉ is cystine; and aa₁₀ is leucine, andretromers, truncations, extensions, combinations, fusions, andderivatives thereof, the peptide having antimicrobial activity. Withinone embodiment the amino acid core sequence further contains the aminoacid sequence aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆, and wherein aa₁₁ isselected from the group consisting of aspartic acid, glutamic acid,lysine, and glycine; aa-₁₂ is leucine; aa₁₃ is glutamine; aa₁₄ and aa₁₅are alanine; and aa₁₆ is leucine. Within another embodiment the aminoacid core sequence further contains the amino acid sequenceaa₁₇-aa₁₈-aa₁₉, and wherein aa₁₇ is selected from the group consistingof tyrosine, phenylalanine and tryptophan; and aa₁₈ and aa₁₉ areselected from the group consisting of lysine, arginine, and glutamicacid. Within yet another embodiment the amino acid core sequence furthercontains the amino acid aa₂₀ selected from the group consisting oflysine, arginine, and glutamic acid.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide of from 11 to 22 amino acids containing an11 amino acid core sequence:aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁ is theamino-terminus of the amino acid core sequence and is alanine; aa₂ isthreonine; aa₃ and aa₄ are selected from the group consisting of lysineand arginine; aa₅ is asparagine; aa₆ is glycine; aa₇ is selected fromthe group consisting of lysine, arginine, glutamic acid and glycine; aa₆is selected from the group consisting of lysine, arginine and glutamicacid; aa₉ is leucine; aa₁₀ is cystine; and aa₁₁ is leucine, andretromers, truncations, extensions, combinations, fusions, andderivatives thereof, the peptide having antimicrobial activity. Withinone embodiment the amino acid core sequence further contains the aminoacid sequence aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇, and wherein aa₁₂ isselected from the group consisting of aspartic acid, glutamic acid,lysine, and glycine; aa₁₃ is leucine; aa₁₄ is glutamine; aa₁₅ and aa₁₆are alanine; and aa₁₇ is leucine. Within a further embodiment the aminoacid core sequence further contains the amino acid sequenceaa₁₈-aa₁₉-aa₂₀, and wherein aa₁₈ is selected from the group consistingof tyrosine, phenylalanine and tryptophan; and aa₁₉ and aa₂₀ areselected from the group consisting of lysine, arginine, and glutamicacid. Within yet another embodiment the amino acid core sequence furthercontains the amino acid aa₂₁ selected from the group consisting oflysine, arginine, and glutamic acid.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide consisting of PMP-1 (Sequence No. 2), andtruncations, retromers, extensions, combinations and fusions thereof,and D-isomeric amino acid, retromeric, N-monomethyl-lysine, andfluorinated amino acid derivatives thereof, the peptide havingantimicrobial activity.

Within another aspect of the invention antimicrobial peptides areprovided comprising a peptide consisting of PMP-2 (Sequence No. 1), andtruncations, retromers, extensions, combinations and fusions thereof,and D-isomeric amino acid, retromeric, N-monomethyl-lysine, andfluorinated amino acid derivatives thereof, the peptide havingantimicrobial activity.

The above described antimicrobial peptides can be utilized in a varietyof methods, either alone or in combination with other ingredientsexcipients, against a variety of organisms such as bacteria and fungi.Within certain embodiments the peptide or peptide compositions of thepresent invention can have direct activity against, or, potentiate othermicrobial agents active against agents such as bacteria and fungi.Within related embodiments the peptides can potentiate antimicrobialactivity of leukocytes against organisms such as bacteria and fungi.

Within certain embodiments of the invention, antimicrobial peptidesCS-FBPa (ADSGEGDFLAEGGGVR) and CS-FBbb (EGVNDNEEGFFSA) are explicitlyexcluded from the formula or sequences provided herein.

The peptides and derivative metapeptides of the invention tested to dateexert potent, broad spectrum antimicrobial activities in vitro, exhibitrapid microbicidal activities in vitro, can be used to potentiateconventional antimicrobial agents, to potentiate other antimicrobialpeptides, are active against many organisms that exhibit resistance tomultiple antibiotics, and enhance the antimicrobial functions ofleukocytes. The peptides and derivative metapeptides of the inventioncan be designed to overcome problems of toxicity, immunogenicity, andshortness of duration of effectiveness due to biodegradation, retainingactivity in plasma and serum, since they are based upon naturalantimicrobial peptides that have lower inherent mammalian celltoxicities than conventional antimicrobial peptides. The peptides andderivative metapeptides of the invention also are linear, and have a lowmolecular mass, reducing the likelihood of producing immunogeniceffects, since small linear peptides have a reduced likelihood of beingimmunogenic as compared with larger parent proteins. Many peptidedesigns are inherently resistant to proteolytic degradation, and exhibitstability in temperatures as high as 80° C., and in extremes ofalkalinity and acidity, ranging from pH 2 to pH 10, for example.Substitutions of D- or other unusual amino acids into the peptidetemplates and derivative metapeptide design templates and theirsubsequent iterations may also increase their degradation timesignificantly, extending their half-life. Furthermore, these peptidesare quite amenable to chemical synthesis and recombinant DNA expressiontechniques, facilitating their production in quantities necessary foruse and evaluation in vitro, and eventual therapeutic applications.

These and other aspects and advantages of the invention will becomeapparent from the following detailed description, the accompanyingdrawings and sequence listing, which illustrate by way of example thefeatures of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the design of RP-1, Sequence No. 3, from modelingof a microbicidal domain of PMP-2 (Sequence No. 1);

FIG. 2A is a three-dimensional graph of the antimicrobial spectra ofRP-1, Sequence No. 3, in vitro (radial diffusion assay);

FIG. 2B is a three-dimensional graph of the antimicrobial spectra ofRP-13, Sequence No. 14, in vitro (radial diffusion assay);

FIG. 3 is a flow chart illustrating the method for developing the novelantimicrobial peptides according to the principles of the invention;

FIG. 4 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention againstStaphylococcus aureus in a pharmaceutically acceptable carrier;

FIG. 5 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention againstStaphylococcus aureus in another pharmaceutically acceptable carrier;

FIG. 6 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention againstStaphylococcus aureus in another pharmaceutically acceptable carrier;

FIG. 7 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention againstStaphylococcus aureus in another pharmaceutically acceptable carrier;

FIG. 8 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention against Salmonellatyphimurium in a pharmaceutically acceptable carrier;

FIG. 9 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention against Salmonellatyphimurium in another pharmaceutically acceptable carrier;

FIG. 10 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention against Salmonellatyphimurium in another pharmaceutically acceptable carrier;

FIG. 11 is a two-dimensional graph of the antimicrobial spectra of themean activity of peptides according to the invention against Salmonellatyphimurium in another pharmaceutically acceptable carrier;

FIG. 12 is a chart of the primary structure of PMP-2, showingderivatives thereof;

FIG. 13 is a chart of the chemotactic index for rabbit PMP-2 (rPMP-2),for various organisms;

FIG. 14 is a flow chart for identifying and evaluating activeantimicrobial domains for modeling of peptides according to theinvention;

FIG. 15 is a chart of structural motifs in PMP-2, in which “S” indicates“sheet”, “T” indicates “turn”, and “H” indicates “helix”;

FIG. 16 is a diagram of the structure of RP-1;

FIG. 17 is a helical wheel diagram of RP-1;

FIG. 18 is a diagram of the structure of RP-13;

FIG. 19 is a helical wheel diagram of RP-13;

FIG. 20 is a summary of the RP-1 in vitro spectrum of activity andpotency;

FIG. 21 is a summary of the RP-8 in vitro spectrum of activity andpotency;

FIG. 22 is a summary of the RP-11 in vitro spectrum of activity andpotency;

FIG. 23 is a summary of the RP-13 in vitro spectrum of activity andpotency;

FIG. 24 is a summary of the in vitro spectra of activity, potency andtoxicity of the RP peptides at pH 7.2; and

FIG. 25 is a summary of the in vitro spectra of activity, potency andtoxicity of the RP peptides at pH 5.5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

While natural antimicrobial peptides can be useful in combatingpathogens exhibiting resistance to multiple antibiotics, eitherindependently or in combination with antibiotic regimens or otherantimicrobial peptides, conventional antimicrobial peptides haveheretofore been viewed as being undesirably toxic, immunogenic, and/orshort-lived.

Platelets contain potent antimicrobial peptides, termed plateletmicrobicidal proteins (PMPs). Our preliminary data support the conceptthat PMPs play a key role in platelet antimicrobial functions, and,therefore, in antimicrobial host defense. PMPs are locally released fromplatelets stimulated with microorganisms or agonists present at sites ofendovascular infection. In vitro, PMPs exert rapid, potent microbicidalactions against a broad spectrum of relevant hematogenous pathogens,including Staphylococcus aureus and Candida albicans. Furthermore,organisms resistant to PMPs cause more severe infections in animalmodels than genetically-related counterparts. These facts demonstratethat PMPs are integral to antimicrobial host defense.

Our preliminary evidence indicates PMPs are released into the vascularcompartment to act in antimicrobial host defense. Therefore, PMPs likelyhave structures which optimize antimicrobial activity, withoutconcomitant mammalian cell toxicity. This distinguishes PMPs fromneutrophil defensins, which arc cytotoxic when released, and lose potentantimicrobial activity in this setting. Additionally, PMPs differ inmass and composition from cytotoxic defensins. PMPs exhibit potentantimicrobial activities against pathogens that are resistant todefensin hNP-1, and PMP mechanisms of action are distinct from that ofhNP-1. Furthermore, PMPs exert significantly less cytotoxic effect onhuman vascular endothelial cells or erythrocytes as compared with hNP-1.These facts suggest PMPs have structure-function correlates thatoptimize antimicrobial activity relative to toxicity.

In addition to their direct microbicidal properties, it is highly likelythat PMPs amplify multiple antimicrobial activities of neutrophils. Ourpreliminary data reveal that rabbit PMP-2 (Sequence No. 1) exhibits aC-X-C motif similar to those present in platelet factor-4 andinterleukin-8 This determinant is a principal hallmark of α-chemokinesthat potentiate crucial neutrophil functions such as phagocytosis,chemotaxis, and oxidative burst. Our initial data suggest PMPs amplifyin vitro phagocytosis and intracellular killing of S. aureus by rabbitneutrophils. Furthermore, PMP-2 (Sequence No. 1) exerts enhancedmicrobicidal activities under conditions of pH that exist in theneutrophil phagolysosome. These findings strongly suggest that certainPMPs may augment crucial neutrophil antimicrobial functions.

Our preliminary data provide a basis for our central discovery that PMPshave specific, independent determinants responsible for directantimicrobial activities and potentiation of neutrophil antimicrobialfunctions. We have shown that these determinants can be isolated,optimized, and utilized to design novel mosaic peptides with selectiveantimicrobial properties. With a goal of designing novel therapeuticsthat have potent and selective antimicrobial functions and low toxicity,optimizing the activities of the distinct structural determinants insuch peptides is essential. Our studies on PMP-2 (Sequence No. 1) arestrategically based on our preliminary data: i) it or a precursor isreleased from platelets exposed to agonists present at local sites ofendovascular infection; ii) it exerts potent microbicidal activity vs.relevant pathogens in vitro; iii) it exhibits a C-terminal domainanalogous to known antimicrobial peptides; iv) it has an N-terminalC-X-C motif related to immunomodulatory α-chemokines; and v) it exertssignificantly greater microbicidal activity at pH 5.5 vs. 7.2,suggesting it has enhanced and/or discriminative activity in neutrophilacidic phagolysosomes.

PMPs represent a unique opportunity to delineate structural determinantsthat likely govern discriminative antimicrobial host defense. Mosaicpeptides discovered may also lead to development of novel anti-infectiveagents with selective or enhanced microbicidal and/or immunomodulatoryactivities against antibiotic-resistant pathogens. Thus, these peptideswill additionally significantly advance our understanding of moleculesthat are likely central to host defense against infection, and mayreveal important new strategies to potentiate antimicrobial hostdefense.

It is clear that platelets respond rapidly and are numericallysignificant at sites of endovascular infection, including infectiveendocarditis, suppurative thrombophlebitis, mycotic aneurysm, septicendarteritis, catheter and dialysis access site infections, andinfections of vascular devices. We reason that platelet degranulation(e.g. PMP release) following sequestration of microorganisms likelyproduces potent and direct antimicrobial activities, and facilitatesneutrophil antimicrobial functions.

The likelihood that platelets and PMPs play a crucial role inantimicrobial host defense in these and other settings has beendemonstrated by the following facts: i) platelets are early andpredominant cells at sites of microbial infection of vascularendothelium; ii) platelets target and internalize microbial pathogens;iii) platelets release microbicidal PMPs when stimulated withmicroorganisms or agonists integral to infection in vitro; iv) PMPsexert rapid and potent microbicidal activity against a broad spectrum ofpathogens in vitro; v) PMPs exhibit structural motifs similar toα-chemokines that potentiate crucial neutrophil antimicrobial functionssuch as chemotaxis and oxidative burst; vi) a broad spectrum ofmicrobial pathogens are damaged or killed by activated platelets; vii)thrombocytopenia increases susceptibility to and severity of diversetypes of infections; and viii) mutant PMP-susceptible pathogens are lessvirulent in vivo as compared with PMP-resistant counterpart strains.Collectively, these facts suggest platelets are key to antimicrobialhost defense, particularly through release of PMPs.

Interaction with neutrophils and monocytes provides an additionalmechanism by which platelets and PMPs likely augment antimicrobial hostdefense. Platelets activated by microbes or other agonists releasechemotactic stimuli for neutrophils and monocytes. Most important amongthese are the C-X-C chemokine platelet factor-4 (PF-4), plateletactivating factor (PAF), or platelet derived growth factor (PDGF). Asubcutaneous injection of PF-4 or PDGF rapidly promotes neutrophilinfiltration in animal models. Intravenous injection of PAF into animalscauses eosinophil infiltration into peribronchiolar tissues. Supportiveof our discovery is the fact that PF-4 potentiates both neutrophilchemotaxis and microbicidal activity in vitro. The fact that PMP-2(Sequence No. 1) is related to human platelet factor-4 strongly suggestsPMP-2 (Sequence No. 1) shares these functions. Additionally, Jungi etal. found that monocytes and neutrophils avidly bind to activatedplatelets, but not to resting platelets. This interaction is mediated byP-selectin, GPIIb/IIIa, and/or thrombospondin expressed on activatedplatelets. Molecules generated from activated monocytes or neutrophilscounter-activate platelets. For example, neutrophil superoxide,peroxides, halides, and myeloperoxidase promote platelet degranulation.

Important to our discovery, platelet factor-4 amplifies neutrophilfungicidal activity in vitro. Serotonin and TXA₂ released from plateletdense granules increase neutrophil adherence to vascular endothelialcells in vitro. Monocyte-derived IL-6 induces cytotoxicity of humanplatelets to Schistosoma mansoni larvae in vitro. Most recently,Christin et al. have demonstrated that platelets and neutrophils actsynergistically in vitro to damage and kill Aspergillus. Collectively,these facts demonstrate that a relevant interplay exists linkingplatelet activation and degranulation with leukocyte activation inantimicrobial host defense. We reason that PMP-2 (Sequence No. 1)release from activated platelets is significantly involved in both therecruitment of neutrophils, and amplification of their antimicrobialfunctions.

Platelet antimicrobial functions are likely associated with release ofpotent antimicrobial peptides. In 1887, Fodor described the heat-stablebactericidal activity of serum, termed β-lysin, distinguished fromheat-labile α-lysin complement proteins. Gengou showed that β-lysinbactericidal activity in serum was derived from cells involved in theclotting of blood. Hirsch later reported that platelets, not otherleukocytes, reconstitute the bactericidal activity of platelet-freerabbit serum. Others have isolated cationic β-lysins from rabbit serumthat are bactericidal against S. aureus or B. subtilis. Tew et. al. andDankert et. al. showed that β-lysins and platelet associatedbactericidal substances (PABS) were released from rabbit plateletsstimulated with thrombin. Notably, Darveau et al. studied peptidesrelated to human platelet factor-4 (PF-4) with antimicrobial capacity.The peptides disclosed herein differ both in origin and strategicmodeling from these prior molecules, although some specific similaritiesexist. As detailed below, we have now isolated and characterized rabbitand human PMPs that likely significantly contribute to the antimicrobialeffects of platelets.

Evidence is mounting in support of our discovery that platelets play anintegral role in antimicrobial host defense. Thrombocytopenia (TCP) hasbeen shown to be a significant, independent predictor of worsenedmorbidity and mortality in patients undergoing cytotoxic chemotherapy.In the absence of neutropenia, TCP correlates with increased incidenceand severity of lobar pneumonia. Anti-platelet agents in theexperimental endocarditis model significantly increase bacteremia andmortality. Moreover, Berney and others have demonstrated thatneutropenia in the setting of normal platelet count does not diminishhost defense against endovascular infection following antibioticprophylaxis in vivo. These findings suggest platelets attenuateinfection in vivo. Our studies in experimental animal modelssubstantiate this concept.

Platelets have indisputable antimicrobial properties, and a compellingbody of evidence strongly supports the concept that they are integralcomponents in antimicrobial host defense. It is highly likely that theantimicrobial effects of platelets involve PMP release in response torelevant agonists present in the setting of infection. Thus, PMPs likelyplay a central role in host defense against infection through directantimicrobial action, and potentiation of neutrophil antimicrobialfunctions.

Human PMPs have structural and functional congruence with rabbit PMPs.Much of our current knowledge about endovascular infections has beenobtained using the experimental rabbit model. This model closelysimulates vascular infections in humans. Thus, characterizing thestructure-activity relationship in rabbit PMP-2 has enabledopportunities to elucidate the role of PMPs and platelets in hostdefense in rabbit or transgenic mouse models, as well as in humans.These long range goals may ultimately contribute to development of newtherapeutic approaches in humans. Additionally, these investigationshave uncovered new insights into host-pathogen relationships, and novelapproaches to the prevention or treatment of infections, particularlythose caused by pathogens which exhibit multiple drug-resistancephenotypes.

We have isolated PMPs from rabbit and human platelets subjected to acidextraction or thrombin stimulation. Thrombin is among the most potentplatelet agonists elaborated in the setting of endovascular infection.Fractions of these preparations were screened for antimicrobial activityby acid-urea and sodium dodecyl sulfate polyacrylamide gelelectrophoresis. All active fractions contained small, and cationic(PMPs). We then used reversed-phase high-performance liquidchromatography (RP-HPLC) to purify PMPs to homogeneity. We have sinceisolated a total of seven distinct PMPs from rabbit platelets. Five PMPsare recovered from acid-extracted rabbit platelets, and two distinctPMPs are predominant in thrombin-stimulated rabbit platelets. We haveshown that microorganisms and microbial components are also capable ofstimulating PMP release in vitro. These data indicate PMP release islinked to agonists generated by tissue damage and infection. We andothers have demonstrated that human platelets contain microbicidalpeptides analogous to rabbit PMPs. These results provide evidence forinterspecies conservation of PMPs in mammalian platelets, underscoringtheir likely role(s) in antimicrobial host defense.

Recent evidence implicates platelets and PMPs in host defense againstinfection in vivo, using two complementary approaches. We have examinedthe role of platelets in defense against a PMP-susceptible (PMP^(S))viridans streptococcal strain in experimental infective endocarditis inanimals either with normal platelet counts, or those with selectiveimmune TCP. Thrombocytopenic animals had significantly higherstreptococcal densities in vegetations as compared with theircounterparts with normal platelet counts. Dankert et al. have alsoimplicated platelet-derived molecules as active in host defense againstinfective endocarditis. These data suggest that platelets and PMPs areimportant in limiting the induction and evolution of endovascularinfection.

Complementary to the above approach, we recently demonstrated that, forS. aureus or S. epidermidis, a positive correlation exists betweeninfective endocarditis source and in vitro resistance to tPMP-1. Thesefindings indicate PMP^(S) organisms are less able to propagateendovascular infection in humans as compared with PMP^(R) isolates.Parallel findings suggest Salmonella resistance to defensins correspondswith increased virulence in vivo. Evidence also exists substantiatingthe likelihood that PMPs participate in the observed antimicrobialfunction of platelets in vivo. We have shown that susceptibility totPMP-1 negatively influences the establishment and evolution of S.aureus of C. albicans infection. In the rabbit model, PMP^(S) C.albicans exhibits significantly less proliferation in cardiacvegetations, and dramatically reduced incidence of splenic disseminationas compared with a related PMP^(R) strain. Similarly, we havedemonstrated that in vitro phenotypic resistance to tPMP-1 correlateswith enhanced virulence in experimental endocarditis due to S. aureus.These results suggest that the host defense function of plateletsinvolves PMP elaboration at sites of infection.

We have also shown that PMPs exert relevant and potent microbicidalactivities against bloodborne pathogens in vitro. We have defined themicrobicidal activities of purified PMPs and tPMPs. Nanomolarconcentrations of these peptides exert rapid, potent in vitromicrobicidal effects against S. aureus, S. epidermidis, viridans groupstreptococci, Escherichia coli (1-5 μg/ml), and a variety of otherbacterial pathogens. We have also demonstrated that PMPs and tPMPs arefungicidal in vitro to C. albicans and Cryptococcus neoformans,suggesting their broad antimicrobial spectra. These peptides aremicrobicidal in physiological ranges of pH (5.5 to 8.0), and in thepresence of plasma or serum. Thus, the antimicrobial activities of PMPsobserved in vitro are relevant to conditions known to exist in vivo, asdiscussed below. Furthermore, we have demonstrated that PMPs arereleased from platelets stimulated with agonists present in the settingof endovascular infection, including S. aureus and C. albicans,staphylococcal α-toxin, or thrombin. These findings suggest certain PMPsare released from platelets in response to tissue trauma, solublemediators of inflammation, or pathogens themselves. Therefore, PMPs arelikely to be introduced into the vascular compartment in a localizedmanner to participate in antimicrobial host defense. We reason that PMPshave structural features that optimize their microbicidal activity andinteraction with complementary antimicrobial host defense mechanisms(e.g., neutrophils), without concomitant host cell toxicity. Thus, ourdiscovery is that structural determinants in PMPs significantlyinfluence their microbicidal and/or neutrophil-modulatory activitiesand/or selectivity. The current application is based on derivation ofnovel peptide sequences based in part on those present in one or morePMPs or tPMPs.

PMPs differ in structure from other endogenous antimicrobial peptides.We have used mass spectroscopy to confirm that PMPs range from about 6.0to 9.0 kDa. Compositional analyses reveal that PMPs and tPMPs containhigh proportions of basic amino acids lysine, arginine, and histidine(total content about 25%); this composition is consistent with theircationic charge. Notably, mass, cystine array, and lysine contentdifferentiate PMPs and tPMPs from neutrophil defensins. Additionally,PMPs and tPMPs are distinguished from platelet lysozyme by mass,composition, and antimicrobial activity. Performic acid-oxidizationreveals that PMPs and tPMPs have two to four cystine residues. We havealso found that two cystine residues in PMP-2 are aligned in a C-X-Cmotif, characteristic of α-chemokines that stimulate neutrophilresponse, as discussed below. Together, these findings suggest there arestructural features in PMPs and tPMPs that are integral to their directmicrobicidal activities and/or abilities to influence neutrophilantimicrobial functions, discussed below.

Our preliminary structural data suggest PMPs and tPMPs exhibitsimilarities to and differences from other endogenous antimicrobialpeptides. Similarities of PMPs to other antimicrobial peptides include:i) composition rich in basic amino acids corresponding to cationiccharge, ii) broad antimicrobial spectra in vitro; iii) potentmicrobicidal activity (nanomolar); and iv) disruption of microbialcytoplasmic membranes involved in their mechanisms of action, discussedbelow. In contrast, PMPs and tPMPs have structural characteristics thatclearly distinguish them from other antimicrobial peptides. For example,defensins are 29 to 34 amino acid peptides of about 3 to 4 kDa mass.Similarly, amphibian magainins and insect cecropins range in mass fromabout 2.5 to 4.5 kDa. PMPs and tPMPs are considerably larger (6.0-9.0kDa). Furthermore, neutrophil defensins have three invariate cystineresidue pairs mediating disulfide bridges. These intramolecular bridgesstabilize defensins, conferring their characteristic amphiphilicturn-sheet-helix conformations. Magainins or cecropins lack suchtertiary structure. However, the primary structures of these lattermolecules induce amphiphilic α-helical motifs analogous to those ofdefensins. In comparison, PMPs 1-5 each contain 3-4 cystine residues,similar to defensins, while tPMPs 1 and 2 contain only 2 or 3 cystineresidues, respectively. These findings indicate PMPs have uniquestructural features related to their selective and unique antimicrobialactivities.

PMPs and tPMPs target and disrupt microbial cytoplasmic membranes. Wehave investigated morphologic consequences of rabbit PMP-2 and tPMP-1exposure to bacterial cells, protoplasts, and lipid bilayers in vitrousing transmission electron microscopy (TEM) and biophysical techniques.Rapid cytoplasmic membrane disruption, followed by cell wall swelling,occurs in S. aureus after exposure to 10 μg/ml PMP-2 or tPMP-1 for aslittle as 15-60 min. Ultrastructural damage precedes detectablebactericidal and bacteriolytic effects. Fungal pathogens are likewisedamaged by these peptides in vitro, S. aureus protoplasts exhibitsimilar damage, indicating membrane injury is independent of thepresence of cell wall. We have also demonstrated that PMPs produce theseeffects through a selective mechanism of voltage-dependent membranepermeabilization, as discussed below. These ultrastructural findingssuggest that one primary target of PMP action is the microbialcytoplasmic membrane. It is important to reiterate that PMPs are likelyreleased into the bloodstream in response to infection, such that theypresumably accumulate locally at sites of infection to act directly andindirectly in antimicrobial host defense. This suggests PMP structuraldeterminants optimize microbicidal activity, and minimize hostcytotoxicity, underscoring the importance of understandingstructure-activity relationships in PMPs.

PMPs exhibit structural features likely related to their antimicrobialfunctions. We have used complementary N-termmal sequencing and PCRtechnology to show that PMPs include novel peptides, and peptides notpreviously known to be microbicidal. Thus, our proposed characterizationof the PMP structural determinants that mediate their antimicrobialfunctions has provided information not previously attainable. Severalexciting findings have emanated from our studies of PMP structures.Amino acid sequences of rabbit PMPs 1 and 2 indicate that the initial 24residues present in PMP-1 are identical to those previously known forrabbit rPF-4. Thus, we have tentatively identified PMP-1 as rPF-4,Furthermore, our preliminary sequencing of the majority of the 74 aminoacid residues in native PMP-1 and PMP-2 reveal novel structural dataregarding rPF-4. In addition, our preliminary data indicate that PMP-2is a variant of PMP-1, differing in a glycine-to-arginine substitutionat PMP-2 residue 25. This suggests PMP-2 is a novel microbicidalanalogue of rPF-4. We have found that PMPs-1, -2, and -4 exhibit acystine-variable-cystine (C-X-C) motif characteristic of theα-chemokines integral to neutrophil stimulation. We have givenparticular attention to the C-X-C motifs in PMPs. Clearly, C-X-Cchemokines such as human PF-4 (hPF-4) potentiate neutrophil chemotaxis,phagocytosis, and microbicidal activities in vitro. The fact that PMP-2has the C-X-C motif demonstrates that it potentiates neutrophilantimicrobial functions, in addition to its direct microbicidal action.This rationale underscores the approach we have taken to differentiateeffects of PMP-2 structural determinants on neutrophil antimicrobialactivities as discussed below. In addition to rabbit PMPs, we haveisolated and characterized the structures of analogous human PMPs.Sequence analyses indicate that human PMPs include: hPF-4; connectivetissue activating protein-III (hCTAP-III); thymosin β-4 (hT β-4);platelet basic peptide (hPBP); RANTES (hRANTES); fibrinopeptides A and B(hFP-A and hFP-B; 4.5), and truncations or fragments of these peptides.Like rabbit PMPs, human PMPs exert rapid and potent in vitromicrobicidal activities against S. aureus, E. coli, and C. albicans.Furthermore, several of these peptides (e.g., hPF-4) possess a C-X-Cmotif analogous to rabbit PMP-2. Together, these structural andfunctional similarities indicate close homologies among rabbit and humanPMPs. This evidence further substantiates our rationale to study rabbitPMP-2 as a means of developing novel antimicrobial peptides, and as amodel for future investigation of role(s) of human PMPs in antimicrobialhost defense.

Our recent studies have provided new evidence that PMPs differ inmechanisms of action from those of other antimicrobial peptides. We usedflow cytometry to study the mechanisms of action of PMPs against S.aureus strain pair 6850 (PMP^(S)) and JB-1 (PMP^(R)) in vitro. StrainJB-1 is a menadione auxotroph of parent strain 6850, and has a decreasedtranscytoplasmic membrane potential (ΔΨ). We used the fluorescent probesdioxycarbocyanine (DiOC₅) and propidium iodide (PI) to quantify theeffects of PMP-2 and human defensin NP-1 (hNP-1) on ΔΨ and permeability,respectively. PMP-2 rapidly depolarized, permeabilized, and killed thePMP^(S) strain; this activity was significantly greater at pH 5.5 vs. pH7.2. Depolarization, permeabilization, and killing of the PMP^(R) strainby PMP-2 was significantly less than the PMP^(S) strain. Menadionereconstituted the PMP^(R) strain ΔΨ to a level equivalent to the PMP^(S)strain. This was associated with increased depolarization,permeabilization, and killing of the PMP^(R) strain due to PMP-2.Therefore, the mechanism of PMP-2 action involves rapid, pH-dependentmembrane permeabilization with membrane depolarization. These effectswere different from hNP-1, or the cationic antibacterial agentsprotamine or gentamicin. For example, membrane permeabilization due tohNP-I was equivalent in the PMP^(S) and PMP^(R) strains, and greater atpH 7.2 than at pH 5.5. Collectively, these data suggest PMPs exertmechanisms of action which differ from hNP-1. These findings imply thatspecific structural determinants significantly influence PMPmicrobicidal activities. Similarly, we have recently found that PMPs areactive against Salmonella typhimurium strains resistant to hNP-1. Forexample, parental strain 14028, intrinsically resistant to hNP-1, was assusceptible to PMP-2 as hNP-1 hypersusceptible strains 4252s and 5996s.These data further support the discovery that PMP microbicidalmechanisms differ from hNP-1.

Preliminary data suggest PMP-2 amplifies neutrophil antimicrobialfunctions in vitro. We have initially studied the influence of PMP-2 onin vitro neutrophil phagocytosis and intracellular killing of S. aureus.In our preliminary experiments, a heterologous system was establishedemploying human neutrophils, pooled normal human serum, or crude rabbitPMP-2. Organisms (5×10⁷/ml) were pre-exposed to sub-lethalconcentrations of serum or PMP-2 for 30 minutes, washed, mixed withneutrophils (20:1), and incubated at 37° C. for 2 hours. We observed asignificant increase in phagocytosis of S. aureus when pre-exposed toPMP-2 (mean organisms/neutrophil=11.2), as compared with serum (meanorganisms/neutrophil=6.4) or buffer control (meanorganisms/neutrophil=3.7; P<0.05 for PMP-2 vs. serum or buffer). Toquantify intracellular killing, neutrophils were lysed, and aliquotsquantitatively cultured to enumerate surviving S. aureus cells. Initialresults suggest PMP-2 enhances intracellular killing of S. aureus at the2 hour time point. For example, only 23.8% of PMP-2-exposed cellssurvived, while 64.1% of the serum exposed, and 78.6% of the buffercontrol cells survived at this time point (P<0.05 for PMP-2 vs. serum orbuffer control). These data support our central discovery that PMP-2augments antimicrobial functions of neutrophils. The fact that PMP-2exhibits a C-X-C chemokine domain that likely promotes neutrophilchemotaxis further justifies our rationale that PMP-2 specificdeterminants amplify these neutrophil functions (see below).

PMP-2 exhibits sequences homologous to chemokine and microbicidaldomains. Recent advances in structural analyses have revealed importantnew information regarding structure-activity correlates amongantimicrobial peptides. For example, it is now known that manyantimicrobial peptides are small, cationic, and have amphiphilicα-helical domains. We have compared PMP-2 and known microbicidal peptidesequences to predict structural features that are likely integral toPMP-2 microbicidal activity. These studies revealed that PMP-2 has manyhallmarks of microbicidal peptides, including: 1) periodic amphiphilicdomains; 2) relatively high hydrophobic moment (M_(H)); and 3)charge-clustering. Additionally, we found that PMP-2 possesses a C-X-Cmotif similar to that found in immunomodulatory chemokines. To testwhether we could isolate and differentiate microbicidal domains fromPMP-2, we employed solid-phase F-moc″ chain assembly to synthesize anovel peptide derived from amino acids 46-63 of PMP-2 (PMP-2₄₆₋₆₃)(FX-PMP-2-46-63, Sequence No. 36) with the following sequence:H₂N-ATKKNGRKLCLDLQAAL-COOH. In preliminary structural analyses, we havefound that PMP-2₄₆₋₆₃ (Sequence No. 36) reflects the conformation of thesame domain in native PMP-2 (Sequence No. 1) as is explained furtherbelow. Moreover, PMP-2₄₆₋₆₃ (Sequence No. 36) exerts the selectivemicrobicidal properties characteristic of PMP-2 (Sequence No. 1), thatare significantly amplified at pH 5.5 as compared to pH 7.2. Thus, thestructure-activity relationship in PMP-2₄₆₋₆₃ (Sequence No. 36) mirrorsthat of native PMP-2 (Sequence No. 1).

We have integrated conventional structural analysis with molecularmodeling in our preliminary studies of PMP-2₄₆₋₆₃ (Sequence No. 36).Purification by RP-HPLC reveals that synthetic PMP-2₄₆₋₆₃ (Sequence No.36) elution is consistent with an amphiphilic, cationic peptide. Thefact that PMP-2₄₆₋₆₃ (Sequence No. 36) RP-HPLC elution time is about 8min earlier than PMP-2 (53.5 minutes) under identical conditionscorresponds with reduced hydrophobicity of PMP-2₄₆₋₆₃ (Sequence No. 36).We have confirmed that purified synthetic PMP-2₄₆₋₆₃ (Sequence No. 36)has the correct sequence and mass by Edman-degradation and MALDI-TOFmass spectroscopy, respectively (MW=1842.2 Da; predicted=1842.3 Da).These data confirm the feasibility of our proposed approaches: we haveidentified, synthesized, purified, and evaluated a microbicidal domainof PMP-2 (Sequence No. 1).

We have investigated PMP-2₄₆₋₆₃ (Sequence No. 36) secondary conformationby Fourier-transform infrared (FTER) spectroscopy. Multi-scan FTIR wasperformed on PMP-2₄₆₋₆₃ (Sequence No. 36) suspended in 0.01% acetic acidadjusted to pH 5.5 or 7.2, and with or withoutpalmityl-oleoyl-phosphatidyl-glycerol (POPG in hexachloroisopropanol) assimulation of a prokaryotic lipid membrane. In aqueous solution at pH5.5 or 7.2, these preliminary studies revealed a strong peak at 1629cm⁻¹ indicating that PMP-2₄₆₋₆₃ (Sequence No. 36) exists in a β-sheetconformation. However, in POPG, PMP-2₄₆₋₆₃ (Sequence No. 36) undergoesdramatic conformation shift to a (β-turn/hairpin structure, exhibiting apeak at 1675 cm⁻¹. These results indicate PMP-2₄₆₋₆₃ (Sequence No. 36)likely undergoes a conformational shift when it interacts with thebacterial membrane.

Molecular modeling of PMP-2₄₆₋₆₃ (Sequence No. 36) corroboratesconventional structural analyses. Our preliminary work to model thesolution structure of PMP-2₄₆₋₆₃ (Sequence No. 36) followed a multistepalgorithm designed to predict the conformation of this and otherpeptides. This algorithm uses a serial four-step approach. First,multiple methods (e.g., Chou-Fasman analyses) were employed to seekregions of consensus in the predicted secondary structure. Next, wesearched the Brookhaven protein database for known sequences withhomology to PMP-2₄₆₋₆₃ (Sequence No. 36) (e.g., PF-4). Resultingpeptides were screened, and those lacking consensus secondary structurewere excluded. The remaining peptides were used as templates forPMP-2₄₆₋₆₃ (Sequence No. 36) backbone trajectory. We then used molecularmechanics to allow each theoretical model PMP-2₄₆₋₆₃ (Sequence No. 36)to relax to corresponding energy minima. Molecular dynamics were thenused to test conformer stability, and the average conformer wasminimized using molecular mechanics. Three conformers of PMP-2₄₆₋₆₃(Sequence No. 36) were initially identified. Two of these were similarsheet-turn-sheet motifs (forming a hairpin loop with C- and N-termini inclose proximity); another was a helical rod. The loop structures wereboth stable in molecular dynamics. The helical rod rapidly collapsed(within 100 psec of simulation time) into a hairpin-like structure andthus was excluded as a model candidate. After minimization, all modelswere similar, with <1 Å rms difference between backbone atoms. Extendedregions of PMP-2₄₆₋₆₃ (Sequence No. 36) were extensively H-bonded. Toconfirm the predictive accuracy of this approach, the first three stepsof this algorithm have been used on selected peptides (15 residues) ofknown conformation. Selected test peptides (with known structures) wereremoved from the Brookhaven database so they would not self-recognize inthe search. Predicted conformers achieved through the above approachclosely resembled experimentally determined structures (rms deviationsof ≦3.5 Å). Thus, our knowledge-based algorithm is reliable, andcorroborates the predictive value of our proposed modeling strategies.

In addition to the knowledge-based algorithm described above, we havealso used energy-based methods, We used systematic and Monte Carlosearches of the Ramachandran angles (φ and ψ) of PMP-2₄₆₋₆₃ (SequenceNo. 36). We found multiple minima on its energy surface, indicating thatseveral conformers were possible. However, the molecular dynamicssimulations demonstrated that the only stable conformer was that of thehairpin loop. In more extended simulations (10 nsec), the peptideoscillated about the hairpin structure as judged by radius of gyrationand Ramachandran angles. The result suggests that the energy barrierbetween conformers is high, and that one conformer predominates or isexclusive. This conformer was the same as that identified by theknowledge-based algorithm described above. In addition, these modelingstudies predicted that the − and C-terminal regions are extendedstructures, with a short helical span central to the peptide. Thesefindings corroborate the β-sheet-turn-β-sheet structure suggested by ourFTIR analyses. Preliminary modeling of PMP-2₄₆₋₆₃ (Sequence No. 36) alsopredicts structural features likely integral to antimicrobial activity.For example, the electrostatic distribution analysis of PMP-2₄₆₋₆₃(Sequence No. 36) indicates that its charge is longitudinally polarized(e.g., strongly cationic C-terminus with a relatively non-chargedN-terminus). Furthermore, PMP-2₄₆₋₆₃ (Sequence No. 36) exhibitssubstantial periodic amphiphilic and hydrophobic clustering. Segregationof charge and hydrophobicity are peptide motifs associated withmicrobicidal activity. Therefore, our preliminary molecular modelingdata reveal a likely structure-activity relationship in the microbicidaldomains of PMP-2 (Sequence No. 1).

It is important to note convergence of the predicted and determinedPMP-2₄₆₋₆₃ (Sequence No. 36) conformations from multiple startingpoints. These findings correspond with FTIR data, indicating thatPMP-2₄₆₋₆₃ (Sequence No. 36) has an anti-parallel strand structure witha short helix forming the connecting region. This agreement among twomodeling algorithms and experimental and FTIR data indicate theconformer identified is the likely solution structure for PMP-2₄₆₋₆₃(Sequence No. 36). The next logical step would be to model PMP-2₄₆₋₆₃(Sequence No. 36) at the surface of a lipid bilayer.

Overall, these data indicate several important features substantiatingthe utility of our proposed molecular modeling strategies. First, wehave gained important insights into the fundamental structure-activityrelationship in PMP-2₄₆₋₆₃ (Sequence No. 36); these consistentlytranslate to PMP-2. Thus, we are poised to define the precise structuraldeterminants in PMP-2 using these methods. Importantly, the predictedPMP-2₄₆₋₆₃ (Sequence No. 36) conformer is not obvious from the primarystructure. Nonetheless, our experimental data corroborate our modelingdata. In addition, the consistency in prediction of the same motifs byboth energy- and knowledge-based strategies suggests this conformationalpreference is genuine. These relationships underscore a major advantageachieved through our integration of conventional structural analysis andmolecular modeling: crucial structure-activity relationships may goundetected if either strategy were to be used exclusively.

A basic peptide is expected to have especially strong interactions withbilayers of acidic phospholipids (e.g., those bearingphosphatidylglycerol and phosphatidylserine head groups). The strongmatrix of net negative charge will act as a cation exchanger for basicpeptides to be investigated in this work. Thus, only the interactionbetween the polar phospholipid head groups and PMP-2 determinants can besimulated to focus computational resources. Lipid environments(bilayers) simulating prokaryotic or eukaryotic membranes can be testedfor interaction with peptides. Two-dimensional grids ofdiacetylphosphatidylglycerol or diacetylphosphatidylserine molecules canbe generated. In the primary simulation, PMP-2 conformers correspondingto local minima (as described above) can be manually docked to the polarsurface of this grid using the SYBYL algorithm DOCK. Molecular mechanicsand molecular dynamics can then be used to estimate the influences ofthe phospholipid charge array on peptide conformation. This will alsoestimate the attraction of a peptide for the phospholipid head group,revealing insights into peptide/target-cell selectivity. Solvent can beassigned as a distance-dependent dielectric function. Initially,phospholipids can be fixed as an immovable aggregate; conformationaltransitions of PMP-2 determinants can then be simulated using molecularmechanics and molecular dynamics as above. In complex secondary models,a phospholipid array can form one wall of a cube comprised of a PMP-2determinant TIP water, and counter ions (e.g., NaCl) when appropriate,and phospholipids initially fixed as before. In other simulations, withand without explicit solvent, flexibility of the polar head groups canbe allowed. In this case, the phospholipids can be anchored by themethyl groups on the acyl chains. We recognize there are limitations tothese simulations, and potential pitfalls can be minimized as pointedout by Jakobsson. These methods have been successfully used tocharacterize peptide-lipid interactions by numerous investigators.

Analytical ultracentrifugation can be used to study hydrodynamic shape,predicted radius of gyration, and therefore, overall fold of thepeptide. As important, centrifugation can ascertain the degree ofself-association of the peptide under the conditions used to assay itsactivity. Self-association may occur through either open association(aggregation increases continually with peptide concentration), orclosed association (the peptide reaches a definite, multimeric state).Knowledge of the aggregation state is essential for completeinterpretation of both the physical and biological data. Experimentalresults can be compared to models; accuracy of the comparisons can bewell within the range to make qualitative differentiations (e.g.,helical rod, folded helix, hairpin, antiparallel β-sheets, random coil).The advantage of analytical ultracentrifugation is that all measurementscome directly from first principles; thus, they do not rely on standards(as do most common analytical techniques). Additionally, only smallquantities of peptide are required, and the technique isnon-destructive.

The following relationship can be employed in these investigations:

s=M(1−vp)/Nf

where s is the sedimentation coefficient, v is the partial specificvolume, p is the density of the solution, N is Avogadro's number, and fis the frictional coefficient. The frictional coefficient (f) is givenby

f=6pηR _(s)

where η is the viscosity and R_(s) is the Stokes radius. The diffusioncoefficient (D) is given by

D=RT/f

where R is the gas constant and T is the temperature in degrees Kelvin.Therefore,

Stokes radii can be obtained by measurement of either sedimentationcoefficient or diffusion coefficient. Both can be determined in thecentrifuge, and in favorable cases, in a single experiment.Self-association can be determined from sedimentation equilibriumexperiments, with the general relationship determined by the equation:M=[2RT1/w⁴(1−vp)][d ln(c)/dr²]. In the absence of self-association, aplot of ln(c) vs. r² is linear. In the presence of self-association, theline can be concave upward. The slope of the line is analyzed and can beused to determine the propensity of the peptide to self-associate usingthe computer program ORIGIN. Because of the large diffusion coefficientsof small peptides, synthetic boundary centerpieces can be used to obtainan initial sharp boundary between peptide and solvent. Band formingcenterpieces can be used depending on preliminary results. The initialconcentrations of peptide may vary between 0.01 and 100 μlg/ml.Diffusion coefficients can be obtained from boundary spreadingexperiments. While these can be obtained from the high-speed syntheticboundary experiments, generally the determinations can be made at lowspeed where sedimentation will be small. Initially, the rotor speed canbe set low, and adjusted upward during the experiment depending on thedetermined concentration gradient. In this way a range of concentrationscan be generated in a single experiment, and any pressure dependenciescan then be identified. Due to the high diffusion coefficient of thepeptides, equilibrium can be attained rapidly. Equilibrium can bedefined as lack of a detectable difference in measurements of theconcentration gradient taken 1 hour apart.

In cases where self-association of peptide is observed in the analyticalultracentrifuge, chromatography can be used to extend analysis to lowerconcentrations which may be more relevant to those used for measurementof biological activity. The total volume of a columnV_(T)=V₀+V_(i)+V_(g), where V₀ is the void volume, V_(i) is the interiorvolume, and V_(g) is the volume of the chromatographic matrix. Theelution volume V_(e)=V₀+KV₁, where K, the distribution function, vanesbetween 0 and 1. The advantage of gel permeation chromatography is theease of use, less interference from buffers, and the lowerconcentrations of peptide that can be analyzed. The disadvantage is thatthe column must be calibrated with standards of known Stokes radii.Guided by our initial experiments, Sephadex GI0, G15 or G25 (orcorresponding Sepharcyl matrices) can be used as the chromatographicmatrix. Chromatography can be conducted at constant temperature using anautomated fraction collector, and peptide detected by opticalabsorbance. When peptide concentrations are low or the buffers stronglyabsorbing, peptide can be detected by reaction with fluorescamine orother reagents, which we can detect in the femtomolar range. Thecombination of analytical ultracentrifugation and gel permeationchromatography will allow experimental verification of predicted peptideconformations, and detection of any anomalies, such as self-association,that influence interpretation of the spectroscopic and biologicalfindings.

The conformational status of PMP-2 determinants and other peptides canbe determined using circular dichroism (CD) and/or Fourier-transforminfrared (FTIR) spectroscopy as previously described. CD can beprincipally be used to assess helicity, and FTIR has advantages indetermining β-sheet structures. Purified peptides can be solubilized toa concentration of 50 μg/ml in 50 mM NH₄HCO₃. Buffer-subtracted CDspectra (190-250 nm) can be obtained from an average of three 25° scans,using a mean residue ellipticity based on a mean residue mass of 110daltons. Attenuated total reflectance (ATR) crystals of selectedpeptides can be produced by adsorption of 500 picomoles onto aluminizedmylar, coated with nitrocellulose. FTIR spectra can be recorded at anaccelerating voltage of 16 kV at 16000 nanosecond intervals, andanalyzed using peak search software.

We have developed novel tools for studies to examine PMP antimicrobialactivities. As described above, we have recently utilized the rabbitmodel of infective endocarditis to explore the host defense propertiesof platelets and PMPs in vivo. Additionally, we have recently developedpathogen strain pairs that differ in susceptibility to PMPs. Theseorganisms have facilitated our investigations into the mechanisms of PMPaction, and studies to evaluate the role of PMPs and platelets in hostdefense against infection. The panel of organisms we have developedinclude both isogenic S. aureus and C. albicans strain pairs whichdiffer in PMP susceptibility. We generated these strain pairs in twoways. First, we developed PMP-resistant (PMP^(R)) strains fromsusceptible (PMP^(S)) parental strains by serial passage through highconcentrations of PMPs in vitro. We then compared these strains (S.aureus 19^(S)/19^(R) ; C. albicans 36082^(S)/36082^(R)) by restrictionmapping, immunoblotting, and phenotypic characterization in vitro and exvivo. Strains were indistinguishable by these techniques other than inPMP susceptibility. We have also developed a panel of PMP^(R) S. aureusstrains by transposon mutagenesis of PMP^(S) strain ISP479 engineered topossess the transposon Tn 551 in a pI258 vector. We identified a clone(ISP479R) with a stable tPMP-1^(R) phenotype after serial passage inbroth media and rabbit serum (>85% survival after 2 hour exposure to 10μg/ml tPMP-1, vs.<10% survival of the parental strain). The PMP^(R)phenotype in this strain was also stable after in vivo passage in therabbit. EcoRI and NcoI restriction analyses and Southern hybridizationwere used to confirm that ISP479R contained a single Tn 551 insert,localized within the same restriction fragment pre- and post-in vitroand in vivo passage. The related strain iSP479C, cured of the plasmidcontaining the pI258 vector, completes the control organisms in this S.aureus strain panel. We have studied this panel extensively in therabbit model of infective endocarditis. In doing so, we have nowdemonstrated that artificially-induced resistance to PMPs confers asurvival advantage to organisms in the context of endovascular infectionin vivo. Thus, susceptibility to PMPs is undoubtedly a significantparameter in overall antimicrobial host defense. Studies beyond thescope of the current application are under way to define the preciseinfluence of PMP resistance in various animal models. Strain pain suchas these are also crucial to future studies to define mechanisms of PMPaction, and the genetic elements in pathogens that may be responsiblefor resistance to PMPs and/or other antimicrobial peptides. In addition,relevant and well characterized strains available from the American TypeCulture Collection (ATCC) will be important tools with which we canevaluate the potencies of our novel peptides against drug-resistantpathogens.

Designs of Novel Antimicrobial Peptides

Our preliminary data strongly support our central hypotheses: i) PMP-2(Sequence No. 1) exerts direct antimicrobial activities linked to itsspecific structural determinants; ii) PMP-2 (Sequence No. 1) potentiatescrucial antimicrobial functions of neutrophils likely due to structuressuch as C-X-C; iii) structure-activity relationships in PMP-2 (SequenceNo. 1) antimicrobial determinants can be isolated and modeled, enablingdesign of novel peptides and mosaic peptides that achieve highly potentand/or selective antimicrobial activities.

In this regard, a defined set of analogues can be synthesized,characterized, and assessed by the above screens for antimicrobialactivity. These approaches have been used to identify specificstructural determinants in PMP-2 responsible for direct antimicrobialactivities. First, truncated versions of PMP-2 domains have beensynthesized. Next, compositions of these domains can be strategicallyvaried to define the specific determinants responsible for theirantimicrobial activities as described above. Criteria for selection caninclude exceptional antimicrobial activity and/or selectivity.Furthermore, combinatorial peptides can be synthesized at the 0.01 nmolscale by simultaneous peptide synthesis methods.

Systematic peptide truncation can be used to define domain sizeessential for antimicrobial activity. In addition, peptides of reducedchain length may be advantageous as therapeutic agents as compared withlarger proteins: 1) smaller peptides typically have greater distributionvia more efficient diffusion; 2) they are generally less immunogenicthan larger peptides; and 3) shorter peptides tend to be lesssusceptible to proteolytic degradation than comparable larger proteins.Thus truncated analogues of PMP-2 functional domains have beensynthesized, including N-terminal, C-terminal, or dual-terminaltruncations using combinatorial synthesis (e.g., see Sequence Nos. 30,31, 32 and 33).

We have noted that charged, hydrophobic, and aromatic amino acidresidues dramatically influence peptide antimicrobial activities. Due tothis relationship, peptide libraries can be derived from selectedtemplates to vary peptide parameters believed integral to antimicrobialactivity individually or in combination: 1) conformation; 2) chargedensity and periodicity; 3) amphiphilic density and periodicity; 4)hydrophobic moment (M_(H)); 5) mass-to-charge ratio; and 6) terminalorientation,

1. Charge-Conservation, Neutralization, or -Reversal: Antimicrobialpeptide potencies may vary relative to steric properties of chargedamino acids. Alternatively, net charge may dramatically influencepeptide activity. Therefore, charged amino acids can be substituted suchthat overall charge can be conserved, but varied sterically (e.g.,lysine-to-arginine), neutralized (e.g., lysine-to-glycine), reversed(e.g., lysine-to-glutamic acid), or a combination of these approaches.

2. Non-Polar Substitution: Hydrophobic amino acids leucine, alanine,isoleucine, and valine are common residues among antimicrobial peptidesequences. These residues likely have a significant impact onhydrophobic density and mean hydrophobic moment (M_(H)) as they relateto peptide antimicrobial activity. Thus, peptides can be designed withnon-polar substitution (e.g., leucine-to-isoleucine) and/or conversion(e.g., valine-to-glycine) to assess the influence of polarity in aminoacids on antimicrobial activities of PMP-2 structural domains.

3. Aromatic Substitution: Aromatic amino acids such as tyrosine,phenylalanine, and tryptophan directly influence mean hydrophobic momentand hydrophobic density. In addition, their molecular radiisignificantly influence the steric properties of peptides. Theseparameters are believed crucial to peptide microbicidal activity.Therefore peptides derived from PMP-2 structural domains can be assessedwith aromatic substitutions for their antimicrobial activities, such astryptophan-for-tyrosine, and phenylalanine-for-tyrosine scanning.

4. Retromer Peptides: Stereo-specificity likely plays an important rolein peptide-target cell interaction. However, previous studies have shownthat L- and D-isomer peptides are indistinguishable in theirantimicrobial activities. Therefore, selected PMP-2 domains exhibitingpotent or selective antimicrobial activities can be synthesized asretromers, and tested as above to assess the influence of terminalorientation on such activities.

The relative rates of modification of amino acid side chains can provideinformation about accessibility and dynamics of many of the studypeptides. Thus, synthetic analogues of selected peptides found to haveexceptional or unique antimicrobial activities can be studied to furtherdefine their structure-activity relationships, as outlined below, andused to design subsequent peptide iterations.

1. Conformer-Restriction Amino Acid Substitution. An excellent method ofconformational control is to replace selected amino acids in theoriginal peptide with amino acids that will restrict the motion of thepeptide chain. In selected peptides, proline, β-branched, N-methyl,α,β-dehydro (unsaturated bond between the α and β carbons), α,α-dialkyl,and/or D-amino acids can be placed at positions allowed or preferred,where Ramachandran φ and/or ψ torsional angles are compatible with thepredicted peptide backbone trajectories. Both protein and non-proteinamino acids can be introduced in combinatorial synthesis of thepeptides. In this way, use of amino acids with restricted φ and ψ angleswill create more active analogs, since these modifications will favorthe conformer with the desired antimicrobial activity.

2. Disulfide-Bridge Conformer Stabilization. We have used the cystinecross-linking method to verify the predicted conformation of an insectneuropeptide. Precise and restricted geometries of disulfide bonds makeengineering of these crosslinks rigorous tests of peptide conformation.Measurement of the rates of disulfide formation in the reduced peptide,along with comparison of biological activity in oxidized and reducedstates provide additional tests of the predicted conformation andactivity relationship. Therefore, selected peptides can be synthesizedto contain cystine residues in strategic locations to facilitatedisulfide bridge-mediated stabilization of test conformations.Biological activities of the reduced (—SH) or oxidized (cystinecross-linked) peptide can then be measured and compared. The reducedpeptide provides a control on possible perturbations introduced byreplacement of the original amino acid residue by cysteine. If thepredicted tertiary structure is correct, the disulfide cross-linkedpeptide should have an efficacy equal to, or greater than, the originalpeptide. The possibility of greater efficacy arises because thedisulfide link restricts the number of possible conformations of thepeptide, thus increasing the effective concentration of the biologicallyactive conformer.

The quantifiable antimicrobial activities of peptides have beendetermined as described above. Through molecular modeling, qualitativecorrelation among structure and activity can be identified. However, itis also important to ascertain quantitative structure-activityrelationships (QSAR) with robust predictive ability in designedpeptides. Comparative Molecular Field Analysis (CoMFA) is a particularlyuseful tool in this regard. For example, CoMFA techniques have beenapplied to model and design novel HIV protease inhibitors, antibacterialagents, antidepressants, ACE inhibitors, and several other molecules nowrecognized as important therapeutic agents. Highly flexible moleculeshave also been successfully analyzed by CoMFA. Furthermore, data fromthese analyses can be integrated with other physical data (e.g.,octanol/water partitioning to measure hydrophobicity and solvationenergy), to augment predictive power.

Initially, two structure-activity analyses can be performed integratingall measures of antimicrobial activity, Holographic-QSAR (HQSAR)analyses can be used, since this method does not require conformationalinformation. Therefore, rational mosaic peptide design can also beachieved, based on quantitative correlations of peptide primarystructure and antimicrobial activity, as soon as the data set ofbiological properties becomes large enough to rise above background.

As is illustrated in the drawings, the invention is accordingly embodiedin novel, improved antimicrobial peptides designed from unique templatesto act to inhibit or kill microorganisms that are otherwise resistant toexisting antibiotics. Two principal peptides, designated RP-1, SequenceNo. 3, and RP-13, Sequence No. 14, were designed based in part uponmicrobicidal domains from platelet microbial proteins 1 and 2 (PMP-1,Sequence No. 2, and PMP-2, Sequence No. 1) as discussed in Yeaman, M.R., et al., “Purification and in vitro activities of rabbit plateletmicrobicidal proteins,” Infect. Immun. 65:1023-1031, 1997. In turn,these or other microbicidal peptides can also be used as structuraltemplates from which iterative peptides can be modeled and synthesized.These peptides, and derived analogues, may eventually be developed astherapeutic agents to significantly improve treatment of lifethreatening infections in humans due to organisms resistant toconventional antibiotics.

In addition to parameters known to be associated with antimicrobialactivity, specific features have been identified which appear to beintegral to maximal peptide microbicidal activity. These include: 1)conformation; 2) charge density and periodicity; 3) amphiphilic densityand periodicity; 4) hydrophobic moment (MH); 5) mass-to-charge ratio;and 6) terminal orientation.

The present invention applies a model which predicts relativeantimicrobial activity for a given amino acid sequence. This model takesinto account the following published equation for determination of themean hydrophobic moment (M_(H)):

$M_{H} = \frac{\left\lbrack \left\lbrack {{\sum\limits_{n = 1}^{N}{H_{n}{\sin \left( {\delta \; n} \right)}^{2}}} + {H_{n}{\cos \left( {\delta \; n} \right)}^{2}}} \right\rbrack^{1/2} \right.}{N}$

where N is the number of residues, H_(n) is the hydrophobicity of thenth residue, δ is the repeat angle, 100°, and M_(H) is the meanhydrophobic moment. We have modified this equation to integrate α and βparameters, where α is the alpha helicity index (helical fraction), β isthe beta-sheet index (sheet fraction). Use of the variables α and β aredescribed below.

Many cationic microbicidal peptides are known to exhibit amphiphilicα-helical or β-sheet conformation. It is also known that manyantimicrobial peptides possess domains rich in hydrophobic amino acids.The mean hydrophobic moment M_(H) dually assesses these parameters;M_(H) is essentially the amphiphilicity of a peptide in an α-helicaconformation. In previous models, M_(H) and amphiphilicity are among themost predictive parameters of actual antimicrobial activity. Theinventors have additionally recognized that potent microbicidal peptidescontain distinct hydrophobic, amphiphilic and hydrophobic domains. Theabove model has been refined to integrate M_(H) and α-helical or β-sheetconformations in the context of such domains. In this model, peptidemicrobicidal activity (predicted MIC, also P_(MIC)) is inversely relatedto M_(H) and α-helicity such that: P_(MIC)=1/∝=[(M_(H))·(α_(peptide))]where α is equal to the sum of the α helical fractions of the peptide.Similarly, β-sheet peptides will be assessed for P_(MIC) as follows:P_(MIC)=1/∝[(M_(H))·(β_(peptide))], where β_(peptide) is equal to thesum of the β sheet fractions of the peptide. P_(MIC) can be inferredfrom the respective outcome of these models as they apply to a helical,β-sheet, or other peptide conformations. In either case, the lower theP_(MIC), the greater the predicted microbicidal activity. This model hasproven successful in guiding selection of templates used in designingtemplates RP-1, Sequence No. 3, and RP-13, Sequence No. 14, and derivedmetapeptides, discussed further below.

The peptide model has been used according to the principles of theinvention to design RP-1, Sequence No. 3, and RP-13, Sequence No. 14,template peptides from microbicidal domains of PMPs 1 and 2, asillustrated in FIG. 1. These peptides exert rapid (less than 2 hours)and potent (nanomolar concentration) microbicidal activities against aspectrum of pathogens in vitro, many of which are resistant toconventional antibiotics, as is shown in FIGS. 2 a and 2 b, reflectingin three-dimensional graphs the antimicrobial spectra of RP-1, SequenceNo. 3, and RP-13, Sequence No. 14, in vitro (radial diffusion assay).Inocula were 1×10⁶ CFU/ml, and incubation conditions were pH 7.2 (RP-1,Sequence No. 3) or pH 5.5 (RP-13, Sequence No. 14), for 24 hours at 37°C. (Key: EC, E. coli; EF, Ent. faecalis; PA, Ps. aeruginosa; SM, St.mutans; SA, S. aureus (MRSA); SE, S, epidermidis (MRSE); CA, C.albicans; CN, Crypto, neoformans) Moreover, these templates differ insecondary structure (α-helix vs. β-sheet, respectively) as determined byFTIR spectroscopy and molecular modeling, and have differential pHoptima for microbicidal activity (pH 7.2 vs. 5.5, respectively). Thus,the use of peptides derived from PMPs 1 or 2, RP-1 or RP-13, or othertemplates will provide complementary opportunities to examine therelationship among peptide structure, microbicidal activity, pathogenspecificity, mechanism of action, conditions for activity, and mammaliancell toxicity. These data will be incorporated into subsequentiterations of peptide design.

With reference to FIGS. 2A. and 2B, designs for novel microbicidalmetapeptides should maximize peptide parameters believed to be integralto microbicidal activity, as discussed above. Specific design strategiescan include charge substitution, non-polar substitution, aromaticsubstitution, peptide extension or truncation, and use of D-enantiomers,retromer, retroenantiomer, N-^(∈)monomethyl-lysine, or other amino acidsnot normally found in native peptides, or any combination of theseapproaches. In addition, conformer restriction and/or disulfide bridgeconformer stabilization can be used to create designs with specificconformational parameters found to be relevant to derived antimicrobialproperties.

In charge substitution, charged amino acids can be substituted withalternate amino acids such that the overall charge is essentiallyconserved. Examples of interchangeable residues where chargeconservation substitution can be used to create novel peptides arelysine and arginine, or aspartic acid and glutamic acid.

Peptides can also be designed with substituted non-polar residues tostudy this effect on peptide microbicidal activity. Leucine andisoleucine are common examples of hydrophobic amino acids inantimicrobial peptides. Such residues have a significant impact onhydrophobic density and mean hydrophobic moment (M_(H)) as they relateto peptide microbicidal activity.

Peptides with enhanced microbicidal activity and reduced mammalian celltoxicity can also be generated with aromatic substitutions. Aromaticamino acids such as tyrosine, phenylalanine, and tryptophan are believedto influence mean hydrophobic moment as well as hydrophobic density.

Peptide extension or truncation can also be used to model peptidedesigns with strategic modifications. Peptides of reduced chain lengthgenerally exhibit features which may be advantageous as potentialtherapeutic agents as compared with larger proteins: 1) smaller peptidestypically have greater distribution via more efficient diffusion; 2)they are generally less immunogenic than larger peptides; and 3) shorterpeptides tend to be less susceptible to proteolytic degradation thancomparable larger peptides. Selected peptides which exhibit potentmicrobicidal activity can also be synthesized as N-^(∈)monomethyl-lysineand/or D-amino acid analogues. These strategies can be useful toincrease specificity, reduce toxicity, and extend half-life of thesepeptides.

Peptides derived from RP-1, Sequence No. 3, and RP-13, Sequence No. 14,or other natural or novel templates will be suitable in mass to model byenergy based methods. This approach can be used to identify stableconformers, and thus the most likely to retain structures believed toconfer microbicidal function. Phi (φ) and psi (ψ) angles can be assignedsystematically; those incompatible with Ramachandran indices forparticular amino acids can be rejected to speed the search process.Conformer side chains can be rotated to relieve unstable stericconfigurations, and promising conformers can be partially minimizedusing AMBER force-field strategies. Lowest energy conformers can befurther analyzed by molecular dynamics to determine stability. TheBrookhaven data base can also be searched for peptides homologous tothese peptides, which can be used as comparative templates. Side chaincontacts can be relieved and minimized by molecular mechanics, andlowest energy conformations analyzed by molecular dynamics. Data fromthese manipulations can be used to remodel first generation peptides,such as RP-1, Sequence No. 3, RP-13, Sequence No. 14, or other templatepeptides to further enhance their antimicrobial properties, and reducetheir toxicity.

Conformation of peptides can also be significantly influenced bysolvation. Promising peptides identified can be solvated in TIP3 water.Solvent effects on molecular dynamic trajectories can be analyzed, andfree energy perturbations used to assess solvent energies. Selectedsolvents can be seeded with counter ions at various concentrations toinvestigate possible conformational changes in peptides induced by ionicinteractions. Furthermore, antimicrobial peptides likely interact withlipid bilayers. At the junction between the aqueous phase and the lipidbilayer, lipid polar head groups create a unique environment; thisenvironment can produce alterations in peptide conformation. Lipidenvironments (bilayers) simulating bacterial or fungal cytoplasmicmembranes (e.g. phosphotidyl glycerol or ergesterol) can be tested forinteraction with peptides. Two dimensional arrays of polar head groupswill be made and immobilized. A uniform solvation field will be used oneither side to simulate the aqueous and hydrocarbon environments. Thiswill permit examination of the effect of charge array on peptideconformation in relationship to lipid interaction. The environment ofthe lipid bilayer can then be simulated by minimizing the dielectricconstant, and removing distance-dependent terms in dielectric function.Analysis of molecular dynamics can also be conducted to examineinfluence of lipid environments on peptide trajectory.

Comparative molecular field analysis (CoMFA) seeks predictions ofbiological activity from amino acid sequences. CoMFA can be conducted intwo ways. First, all peptides can be equilibrated in a common extendedconformation, and their side chains relaxed. A conventional CoMFA canthen be constructed. This approach takes advantage of the fact thatCoMFA does not appeal to any one mechanism of action, and seekscorrelations between changes in structure and changes in biologicalactivity. Induced folding should be implicit in the CoMFA analysis. In asecond method, each peptide can be modeled in the lowest energyconformer, and conformers can be used to construct potential fields tobe analyzed by CoMFA.

Novel antimicrobial peptides suitable for use within the presentinvention can be synthesized directly, or, developed using combinatorialchemistry libraries (Silen, J, L, A. T. Lu, D. W. Solas, et al.,Antimicrob. Agents and Chemother 42:1447-1453 (1998)). Briefly,combinatorial libraries can be made by using split-and-pool synthesis,as described by Furka et al. (Furka, A., F. Sebestyen, M, Asgedom, etal., J. Pept. Protein Res. 37:487-93 (1991)). For example, beads aredistributed into three reaction vessels, and an amino acid (A, B, or C)is coupled to the beads. The beads are pooled and redistributed to thesame three reaction vessels, where the another amino acid is coupled,resulting in a dipeptide. This creates a set of 2×3 peptides: AA, AB,AC, etc. The process is repeated once more for example, to create a setof 27 tripeptides. A fundamental consequence of this approach is thatthere can be millions of beads used in the synthesis, with each beadcarrying one unique compound that must be screened and identified.

Several approaches can be used to identify the structure of the compoundcarried on an individual bead. The compounds are tethered to the beadsvia UV photo-labile linkers to allow release of the compound for assay(Holmes, C. P., and D. G. Jones, J. Org. Chem. 60:2318-2319 (1995)).Chemical identifier tags that can be detected more efficiently than thelibrary compound that they represent, are added to the beads after eachsynthetic step. Thus each bead carries a record of the synthesis of thecompound also carried on that bead. By “reading” this tag, one candeduce the identity of the compound carried on the bead. Numerous lagsand analytical methods for reading these tags have been developed (Kerr,J. M., S. C. Banville, and R. N. Zuckermann, J. Am, Chem. Soc.115:2529-2531 (1993); Krchnak, V., A. S. Weichsel, D. Cabel, et al.,Pept. Res. 8:198-205 (1995); Needels, M. C., D. G. Jones, E. H. Tate, G.L. et al., Proc. Natl. Acad. Sci. USA (1993)).

Jayawickreme et al. (Jayawickreme, C. K., G. F. Graminski, J. M.Quillan, et al., Proc. Natl. Acad. Sci. USA 91:1614-1618 (1994))presented the first evidence that single-bead activity fromantimicrobial peptides could be detected on acid-cleavable beads in abacterial cell lawn format assay. For sensitive screening the library ofbeads can be manually spread on 105-μm-pore-size polyester mesh(Spectrum) that is subsequently placed on a nitrocellulose membrane(Bio-Rad) resting on 0.4% PBS agarose. Following 30 min of photolysis,the mesh is covered with a layer 0.4% LB agarose containing ˜10⁷ CFU ofB. subtilis and incubated overnight. Compounds with antimicrobialactivity are identified by zones of inhibited growth. Beads located inthe center of the zones are selected for decoding, by manually isolatingthem from the assay plates. The encoded peptide is re-synthesized andantimicrobial activity is confirmed by testing in a standard brothmicrodilution assay against B. subtilis or other target microorganism ofinterest. Antimicrobial peptides desirably have minimum inhibitoryconcentrations against target microorganisms of <32 μg/ml.

Promising metapeptides and their iterations designed from microbicidaltemplates such as those described (e.g., RP-1, Sequence No. 3, andRP-13, Sequence No. 14) above can be synthesized by solid-phase Fmoc(9-fluorenyl-methyloxycarbonyl) chemistry. The method is established,and has been extensively used in production of antimicrobial peptides.Preliminary amino acid analysis can be performed on samples of materialto estimate overall coupling efficiency and to confirm peptidecomposition. Peptides can be cleaved and deprotected, and purified bygel filtration (BioGel P-10) and reverse phase-HPLC (RP-HPLC). Thislatter instrument can be equipped with a variety of columns includingC-4, C-8, and C-18 silica-based reversed phases (Vydac), and syntheticphases such as PRP-300 (Hamilton) used to purify crude peptides on apreparative scale. Following purification, peptides can be quantitatedby amino acid analysis utilizing the Pico Tag system. Molecular mass ofeach peptide can then be confirmed by fast atom bombardment orelectrospray mass spectrometry. Fourier-Transform infrared spectroscopy(FTIR) and molecular modeling can then be used to verify the predictedsecondary structure of synthetic peptides. In some cases, conformationalstudies can be performed using analytical ultra-centrifugation usingestablished Stokes radius (radius of gyration) predictions to detectpossible peptide-peptide interactions. This approach to peptideproduction and structural confirmation is highly efficient: a peptidecan be synthesized, purified, and verified for sequence and conformationover a ten-day period.

Peptides are tested for antimicrobial potency and spectra against apanel of bacterial and fungal pathogens representing multipleantibiotic-resistance. This panel will include both clinical isolates aswell as genetically-defined laboratory strains which exhibit MIC valuesconsidered resistant to respective antibiotics. Comparative controlorganisms to those assembled are summarized in Table 1 below.

TABLE 1 Antibiotic Resistance Phenotype Control Organism Strain Bla VanAmg Amb Flu Staphylococus Aureus ATCC R S R N/A N/A 27217 Streptococcuspneumonia ATCC R S R N/A N/A 35088 Enterococcus Faecalis ATCC R R R N/AN/A 47707 Escherichia coli ATCC R S R N/A N/A 43827 Pseudomonasaeruginosa ATCC R R R N/A N/A 17468 Candida Albicans ATCC N/A N/A N/A SS 36082 Candida Krusei ATCC N/A N/A N/A S R 32672 Candida LusitaniaeATCC N/A N/A N/A R R 42720 (Key: R, resistant; S, sensitive; Bla,β-lactams; Van vancomycin; Amg, aminoglycoside; Amb, amphotericin B;Flu, fluconazole.)

A central goal is to correlate peptide structure with function toidentify peptides with potent activity and reduced toxicity. Criteriafor success are two- to ten-fold increases in potency as compared withtemplates RP-1, Sequence No. 3, or RP-13, Sequence No. 14. In thisregard, it is advantageous to assess the microbiostatic and themicrobicidal activities of peptides, and to correlate these activitieswith mammalian cell toxicity. For all assays, organisms are cultured tologarithmic-phase per NCCLS guidelines.

We have used the agar radial diffusion assay to determine antimicrobialactivities of proteins against microbial pathogens in vitro. One millioncolony forming units are mixed into 10 ml (i.e., 1×10⁵ CFU/ml) of melted1% agarose (in 10 mM NaHPO₄ and cooled to 42° C.) containing minimalnutrient and adjusted to either pH 5.5 or pH 7.2. The agar is solidifiedin culture dishes, and sample wells are formed. Peptides at variousconcentrations are dissolved in 10 μl of 0.01% acetic acid buffer (pH5.5 or 7.2), loaded into individual wells, and incubated at 37° C. forthree hours. The plate is then overlayed with 1% agarose containingnutrients and incubated (37° C., for at least 24 hours). Peptidespurified by RP-HPLC lacking antimicrobial activity are tested inparallel as controls. Zones of inhibition are measured to quantifyantimicrobial activity. This assay will not distinguish betweenmicrobicidal and microbiostatic actions, but is highly sensitive topeptides with one or both functions.

Minimum inhibitory (MIC) and microbicidal concentration (MMC) assays canalso be performed, and may include a microvolume assay which is used toquantitatively screen peptides for antimicrobial activities. In thisassay, suspensions of bacteria or fungi in appropriate media are placedin 100-200 μl final volumes in microtiter plates. Standard (uncoated),poly-L-lysine coated, or otherwise positively charged plates may be usedfor these assays, since cationic peptides may bind to strongly anionicsurfaces. Purified peptides are then serially diluted, descending from100 μl/ml. Organisms are inoculated into wells to a concentration of1×10⁵ CFU/ml, and plates incubated (37° C., for at least 24 hours). Wellturbidities are then assessed visually and by spectrophotometry toquantify growth inhibition versus wells containing no peptide. MMCs arethen determined by quantitative culture of MIC wells exhibiting novisible growth.

Microbicidal kinetics of purified peptides are assessed by resuspendingthe peptides in 0.01% acetic acid buffer (pH 5.5 or 7.2), and organismsare resuspended to a concentration of 1×10⁵ CFU/ml in 50-250 μl ofsterile buffer containing peptide concentrations from 0 to 40 μl/ml.Controls contain buffer alone or non-antimicrobial proteins and organismas above. Mixtures are incubated at 37° C. for up to 48 hours, afterwhich aliquots are quantitatively cultured and incubated for 24 to 48hours. Killing is expressed as decrease in logarithm₁₀ surviving CFU/ml.The limit of sensitivity in microbicidal assays is considered to be a 1log reduction in viable cells.

Flow cytometry can also be used to examine kinetics and mechanisms ofthe action of the peptides on bacterial membrane integrity andenergetics. Peptides which differ in activity or specificity for theirability to depolarize and/or permeabilize microbial membranes can alsobe compared by analysis of membrane depolarization, andpermeabilization. DiOC₅ is a charged lipophilic dye which partitionsinto the cytoplasm, and is dependent on intact Δψ for intracellularretention. Organisms prepared as above are labeled in darkness for 30minutes at about 20° C. in PBS containing 0.05 μM DiOC₅. Organisms areresuspended to a concentration of 5×10⁸ CFU/ml in K⁺MEM containing anindividual peptide, and incubated at 37° C. For flow cytometry,organisms are washed, sonicated, counted, and resuspended in K⁺MEMbuffer. Reductions in mean DiOC₅ fluorescence relative to controls areinterpreted to represent loss of DiOC_(s), indicating membranedepolarization. Positive control cells exposed to valinomycin, as wellas control cells not exposed to any peptides, are analyzed for DiOC₅fluorescence in parallel.

Propidium iodide is excluded from cells with normal membrane integrity,but enters cells permealized to molecules ≧2 nm in diameter, and can bestimulated to emit fluorescence at >620 nm. Organisms prepared as aboveare resuspended to a concentration of 5×10⁸ CFU/ml in K⁺ MEM containinga selected peptide, and incubated for pre-selected times (ranging fromzero up to about 120 minutes) at 37° C. Cells are washed in fresh K⁺MEM,sonicated, counted, and resuspended in KIMEM buffer containing 20 μMpropidium iodide. Control cells exposed to ethanol (positive control forpermeabilization) are assessed for propidium iodide uptake in parallel.Increases in mean propidium iodide fluorescence relative to controlcells are interpreted to indicate increases in permeability.

Erythrocyte permeabilizing and hemolytic activities of peptidesexhibiting potent microbicidal activity are also studied as indicatorsof potential in vivo toxicity. Four-percent (vol/vol) of washed humanerythrocytes (in PBS alone, or in PBS plus 10% heat-inactivated PNHS areincubated with selected peptides ranging in concentration up to 100times greater than geometric mean MICs. After 24 hours of incubation at37° C., erythrocyte permeabilization and hemolysis are determinedspectrophotometrically. Permeabilization and hemolysis will be comparedto buffers alone, and with a triton X-100 control (100% hemolysis).

Endothelial cell injury due to peptides is measured using a standardchromium (⁵¹Cr) release assay, described in Filler, S. G., et al.,“Candida stimulates endothelial cell eicosanoid production” J InfectDis. 1991, 164:928-935; Filler, S. G., et al., “Mechanisms by whichCandida albicans stimulates endothelial cell prostaglandin synthesis”Infect Immun. 1994, 62:1064-1069; Filler, S. G., et al., “Penetrationand damage of endothelial cells by Candida albicans” Infect Immun. 1995,63:976-983. Briefly, endothelial cells in 96 well tissue culture platesare incubated with Na⁵¹CrO₄ overnight. The following day, theunincorporated isotope tracer is removed by rinsing, and peptides in0.01% acetic acid buffer are added to the endothelial cells. Controlwells are exposed to buffer alone. After a predetermined incubationperiod, the medium is aspirated and the amount of ⁵¹Cr released into themedium is measured by scintillation. This approach facilitates toxicityscreening of multiple peptides simultaneously, and minimizes the amountof peptide necessary for assessment.

Each antimicrobial and toxicity assay described above is performedindependently a minimum of two times, and means±standard error iscalculated for each peptide under varying exposure conditions(concentration or pH) as compared with control samples. Statisticalanalyses of microbicidal data are performed using Student t test orKruskall-Wallis rank sum analysis for non-parametric data, and correctedfor multiple comparisons as appropriate.

Leukocyte Potentiating Antimicrobial Peptides

PMP-2 structural determinants also have effects on neutrophilantimicrobial functions. The antimicrobial roles of neutrophils arecritically linked to their capacity to respond to stimuli generated atsites of infection, undergo directed migration toward these sites, andexecute antimicrobial functions once there. Chemokines exhibiting thecystine-variable-cystine motif (C-X-C) are potent stimulants of theseresponses. Peptides that selectively amplify this activity are not onlyintegral to antimicrobial host defense, but they are also reasonabletargets for study as novel anti-infective agents. PMP-2 exhibits anN-terminal C-X-C motif Furthermore, our preliminary structural dataindicate that PMP-2 is an analogue of PF-4, a C-X-C chemokine known toamplify neutrophil chemotaxis and oxidative burst. Moreover, ourpreliminary studies suggest that PMP-2 amplifies in vitro neutrophilphagocytosis and intracellular killing of S. aureus. Additionally, PMP-2exerts significantly greater microbicidal activity under conditions ofpH consistent with those known to exist in the neutrophil acidicphagolysosome (e.g., pH 5.5). Based on these rationale, we hypothesizethat PMP-2 has structural determinants that potentiate neutrophilfunctions crucial to antimicrobial host defense.

Alpha-chemokines such as PF-4 and IL-8 are critical in amplifying thehost inflammatory responses to infection. For instance, theconcentration of macrophage-derived IL-8 is directly correlated withneutrophil number in human pleural effusions. Furthermore, inhibition ofIL-8 by monoclonal Abs prevents neutrophil influx inlipopolysaccharide-induced pleuritis in rabbit models. These C-X-Cchemokines also potentiate the microbicidal function of neutrophils.Nibbering et. al. have noted that IL-8 potentiates non-oxidativeintracellular killing of Mycobacterium fortuitum by human granulocytes.Additionally, IL-8 enhances in vitro neutrophil microbicidal activityagainst Candida albicans. Petersen et, al. have recently shown humanPF-4 acts along with other chemokines to potentiate neutrophilantimicrobial response. We have determined that rabbit PMP-2 possesses aC-X-C motif homologous to that found in α-chemokines We have alsodetermined that at least two microbicidal peptides from human platelets,hPF-4 and hCTAP-III, also contain this motif hPF-4 is chemotactic forneutrophils, and enhances neutrophil phagocytosis of microorganisms invitro. An additional mechanism through which PMP-2 may augmentneutrophil microbicidal function lies in its enhanced microbicidalactivities acidic pH, such as exist in the neutrophil phagolysosome.Thus, PMP-2 on the microorganism surface may have greater microbicidalactivity once ingested by the neutrophil. Results from our preliminary.studies are consistent with this discovery. From these perspectives,PMP-2 likely potentiates critical antimicrobial functions of neutrophilsin addition to exerting direct antimicrobial activities.

PMP-2 contains a C-X-C motif, and exerts significantly greatermicrobicidal activity under conditions of pH that exist in the acidicphagolysosome of the neutrophil (e.g. pH 5.5). The dominantthrombin-induced PMP (tPMP-1) tacks the C-X-C motif, and exhibitsdiminished microbicidal activity at pH 5.5. Therefore, evaluation ofPMP-2 domain influences on neutrophil function can permit assessment ofthe importance of both the C-X-C motif (±the E-L-R motif; discussedbelow) in the context of overall primary structure, as well as therelationship of pH and microbicidal activity in enhancing neutrophilantimicrobial functions. Of interest is the influence of PMP-2 domainson neutrophil antimicrobial function in vitro and the quantification oftheir effects on neutrophil chemotaxis, phagocytosis intracellularkilling of microorganisms. PMP-2 domains found to amplify phagocytosisor intracellular killing by neutrophils can be assessed for theirinfluence on oxidative burst in neutrophils. PMP-2 domain-mediatedoxidative potentiation can be differentiated from non-oxidativeneutrophil potentiation in this manner. Results from these studies canbe used to guide subsequent experiments to define the specificity ofPMP-2 determinants in augmenting neutrophil antimicrobial functions.

A central goal of the differentiation of the effects of PMP-2 structuraldeterminants on neutrophil antimicrobial functions is the comparison ofPMP-2 domains that influence neutrophil microbicidal action with thosethat confer direct antimicrobial functions. The fact that C-X-Cchemokines potentiate neutrophil antimicrobial functions has been wellestablished. Yet, how this occurs has been complicated by the recentdiscovery of two distinct C-X-C receptors, CXCRI and CXCR2, co-expressedon mammalian neutrophils. Each of these receptors is a 7-transmembranedomain protein functionally coupled to G protein activation. Althoughboth receptors bind IL-8 avidly, they differ in selectivity for otherC-X-C chemokines, such as PF-4. The principal difference in structurebetween IL-8 and PF-4 is a N-terminal glutamic acid-leucine-arginine(E-L-R) motif that immediately precedes the initial cystine residue inthe C-X-C motif of IL-8. Interestingly, IL-8 is considered the onlyrelevant ligand for CXCRI. Activation of neutrophils via the CXCRIreceptor also requires presence of a basic amino acid determinant in thesixth position after the second C-X-C motif cysteine residue. IL-8exhibits this determinant, but PF-4 does not. This fact has beensuggested as a principal mediator of CXCRI specificity. Based on thefact that PMP-2 exhibits an N-terminal C-X-C motif homologous with thatof IL-8, and that it is an analogue of rPF-4 known to induce neutrophilchemotactic response, we hypothesize that PMP-2 stimulates neutrophilchemotaxis. However, PMP-2 lacks the E-L-R and the basic sixth-positionmotifs (PMP-2 has leucine in residue position 21) linked to CXCRIspecificity. Thus, we further hypothesize that PMP-2 stimulation ofneutrophil chemotaxis specifically occurs through the CXCR2 receptor.Thus, synthetic domains of PMP-2 can be constructed that do or do nothave the E-L-R and/or basic sixth residue motifs believed to interactspecifically with the CXCRI receptor. This approach can define whetherPMP-2 domains or other peptides influence neutrophil antimicrobialfunction via the CXCR1 or CXCR2 receptor. Rabbit and human neutrophilresponses to PMP-2 structural domains ±E-L-R and/or basic residue motifscan be compared to define species specificity of these peptides. Inaddition to defining the specificity with which PMP-2 determinantsinfluence crucial neutrophil antimicrobial functions, such in vitrostudies can facilitate future investigations to define the role of PMPsin host defense in vivo. Since such in vivo studies cannot initially beperformed in humans, PMP-2 can yield information applicable to thesefuture studies using rabbit models of infection.

In investigation of the influence and specificity of PMP-2 domainpeptides on neutrophil chemotaxis in vitro, rabbit neutrophils can beisolated from fresh whole blood and labeled with ⁵¹Cr. To conservepeptide, a micro-well assay can be used that is modified from thosedescribed by Boyden and Schroder. In these assays, 2.5×10⁶ neutrophilsare placed in the upper compartment of a chemotaxis microchamber(Neuroprobe), separated from a lower chamber by a membrane having a 3 μmpore size. Purified peptide (1-5 μg) in 2 mM acetate buffer is thenplaced in the lower compartments. Appropriate positive controls assessedin parallel can be N-f-met-leu-phe, IL-8, rabbit or human PF-4, or PMP-2in the same buffer, or buffer alone. Chambers are then incubated for 1hr at 37° C. in 5% C0₂. Upper chambers are removed, rinsed extensively,and counted by scintillation relative to respective controls:lower-compartment fluid; rinses of the upper or lower compartment; and acontrol for neutrophil specific activity. The number of neutrophilspresent in the upper and lower compartments can be interpreted in thecontext of these controls. Mean standard error of the mean (SEM) numbersof cells in each compartment can be determined and compared for eachstimulus. Each condition is tested in triplicate, including bothexperimental and control peptides.

If a peptide increases migration of neutrophils, chemokinesis can bedifferentiated from chemotaxis using a modification of the checkerboardassay described by Cutler. For these studies, chemotactic gradients canbe eliminated by placing purified peptide in the upper compartmentsalong with neutrophils. These assays are performed under incubationconditions (<2 hr) to prevent peptide diffusion beyond specifiedcompartments. This could cause neutrophils responding chemotacticajly tocease or reverse direction, artificially reducing peptide-mediatedneutrophil chemotaxis. Neutrophil migration is assessed as above. Adecrease in the magnitude of neutrophil migration is interpreted toindicate that the peptide is chemotactic for neutrophils. Alternatively,no change in mean neutrophil migration indicates that the peptideupregulates neutrophil chemokinesis.

Results from chemotaxis studies above can be used to guide subsequentexperiments to define the specificity of PMP-2 determinants inneutrophil modulation, as outlined below:

-   -   1. PMP-2 domains stimulating neutrophil chemotactic response can        be for tested for activity in the presence and absence of        monoclonal Ab directed against the CXCR1 receptor, or CXCR2        receptor, or both Inhibition or reduction of PMP-2 domain        stimulation of neutrophil chemotaxis under these conditions will        define the specificity of this effect to the CXCR1 or CXCR2        receptors, or to a mechanism that is independent of these        receptors (e.g. peptide activity in the presence of both        monoclonal Abs). Additionally, analogues of selected,        antimicrobial peptides can be synthesized with either the E-L-R        or basic sixth residue motifs, or both. Resulting alterations of        CXCR1 vs. CXCR2 peptide specificity in neutrophil chemotaxis        provide further evidence for engineered selectivity of PMP-2        determinants for specific neutrophil chemotactic receptors.    -   2. Likewise, selected PMP-2 domains or other peptides that fail        to prompt neutrophil chemotaxis can be synthesized as analogues        that contain the E-L-R and/or basic sixth residue motifs. The        conversion of an inactive peptide to one that stimulates        neutrophil chemotaxis is interpreted as evidence that it lacks        these specific structural motifs corresponding to its inherent        selectivity in neutrophil stimulation.    -   3. The influence of peptides and/or their analogues described        above on human neutrophils can be assessed. These studies will        lend insights into the specificity of peptide determinants or        analogues on human neutrophils that co-express the CXCR1 and        CXCR2 receptors. Results from these studies can be used to guide        future efforts to create novel therapeutics that exert selective        modulatory effects on human neutrophils.

Additionally, peptide analogues can be achieved using a combinatorialmethod, and therefore highly efficient with regard to both time andexpense. It is important to note that it is also possible that peptideswill act via mechanisms not previously described. This possibilityunderscores a major advantage of the proposed approach, which isintentionally not biased to identify any single specificity. Thus, theproposed approaches may also reveal novel interactions between peptidesand neutrophils.

Flow cytometric Analysis of neutrophil antimicrobial functions in vitrocan be evaluated using contemporary flow cytometry techniques. Use offlow cytometry has the advantages of analyzing the characteristics ofindividual cells, as well as the interactions between a large number ofneutrophils and microorganisms. This methodology facilitates the rapiddifferentiation of subpopulations of neutrophils that have distinctantimicrobial responses. In addition, flow cytometry provides highspecificity and quantitative precision. Flow cytometric experiments canbe performed using a FACScan (Becton Dickinson) device when a singlelaser stimulation is sufficient. When multiple excitation wavelengthsare required, the dual laser FACStar IV (Becton Dickinson) can be used.

Influence of synthetic peptides on microorganism phagocytosis byneutrophils in vitro can be evaluated by multicolor flow cytometry. Thiscan be done from two perspectives: i) effect of microorganism exposureto peptide on subsequent neutrophil phagocytosis; and ii) effect ofpeptide priming of neutrophils on subsequent microorganism phagocytosis.Target organisms in these studies are control strains, and neutrophilstreated with cytochalasin D serve as phagocytosis-negative controls.Microbial cells are fluorescence-labeled by incubation in appropriatemedium containing 20 μM bis-carboxyethyl-carboxyfluoresceinpentaacetoxymethylester (BCECF-AM, Calbiochem). BCECF-AM diffuses intomicroorganisms, where it is cleaved by cytoplasmic esterases to yieldthe membrane-impermeable fluorescent markerbis-carboxyethyl-carboxyfluorescein (BCECF). BCECF is retained by viableorganisms, thus serving as a microorganism-specific label.Alternatively, neutrophils can be labeled in RPMI medium containing 5μg/ml phycoerythrin (PE)-conjugated monoclonal antibody My-7 (CoulterInstruments; 45 min, 20° C.). My-7 is directed against the neutrophilCD-13 surface antigen. Therefore, PE-labeled neutrophils are readilydistinguishable from BCECF-labeled microorganisms. Neither BCECF norMy-7 labeling methods significantly alter microorganism or neutrophilphysiology, respectively, as determined in previous studies. Labeledmicroorganisms are then washed and suspended in 2 mM acetate buffer (pH5.5 or 7.2). Peptide is added to labeled microorganism suspensions toachieve the following conditions: i) final inocula of 10⁶ CFU/ml; andii) final sub-lethal peptide concentrations ranging from 0.5 to 5 μg/ml.To conserve peptides, volumes are 500 μl. Incubation is initiated by theaddition of peptide to the microbial inoculum, and continued at 37° C.At predetermined timepoints (0, 15, 30, 60, and 120 minutes), 100 μlaliquots are washed in RPMI to remove excess peptide Organisms are thenassessed to ensure they have retained the BCECF label following peptideexposure.

For phagocytosis assays, labeled, peptide-exposed microorganisms aremixed with neutrophils in RPMI±10% pooled normal serum (PNRS) to achievea neutrophil-to-target cell ratio of 1:100. Three samples of cellsprepared as above are included in each phagocytosis assay: 1) labeledmicroorganisms in flow buffer alone (control for BCECF label specificityand intensity); 2) labeled neutrophils in flow buffer alone (control forMy-7 label specificity and intensity); and 3) labeled microorganismsmixed with labeled neutrophils. Mixtures will be incubated for 0, 15,30, 60, or 120 min at 37° C. with agitation. To differentiatemicroorganism binding from phagocytosis, mixtures are cooled on ice toprevent further phagocytosis, and texas red conjugated monoclonalantibody directed against respective organisms (e.g., anti-S. Aureusprotein A; ImmunoSys) is added to samples containing neutrophil-organismmixtures. Therefore, fluorescein emission (520 nm) corresponds tophagocytized organisms, while texas red emission (620 nm) specifiesextracellular organisms when stimulated at 460 and 580 nm, respectively.Furthermore, fluorescein and texas red emissions are distinguishablefrom that of phycoerythrin-labeled neutrophils (575 nm). Organisms whichdo not retain the BCECF label are gated out of data in all phagocytosisstudies. We appropriately monitor forward and 90° light scatter tominimize the collection of artifactual data due to cell clumping. Inparallel, 100 μl aliquots are removed and analyzed by flow cytometry todetermine microorganism viability (see below). Additionally, phagocyticassays are performed microscopically to confirm flow cytometric data.These controls allow us to adjust for underestimates in phagocytosisthat may occur via microorganism loss of BCECF due to killing that mayoccur at later time points.

As an alternative approach to differentiating ingested vs.neutrophil-bound organisms, the fluorescence of extracellularmicroorganisms labeled with BCECF can be quenched by crystal violet,while fluorescence of those within neutrophils is unchanged.Additionally, fluorochrome-quenching reagents (Molecular Probes) thatwill de-fluoresce extracellular organisms, or use of fluorochromes withdifferential emission spectra within the neutrophil acidic phagolysosome(e.g., SNARF; Molecular Probes) can also distinguish pathogen bindingvs. phagocytosis.

In order to determine the influence of peptides on intracellular killingof microorganisms by neutrophils, coincident with phagocytosis assaysabove (0, 15, 30, 60, and 120 minutes), 100 μl aliquots from eachphagocytic assay sample can be processed to quantify intracellularkilling. Neutrophils are lysed in cold distilled water and sonication,and microorganism survival assessed by flow cytometry. As above, viablemicroorganisms retain the BCECF label, while killed organisms lose thefluorescent label. Thus, microorganisms released by neutrophil lysis canbe gated into one of two populations based on fluorescence to quantify:i) viable, fluorescent cells, or ii) non-viable, non-fluorescent cells.Interpretation of results in the context of control neutrophil killingof organisms permits comparison of the influence of peptide exposure(either microorganism, or neutrophil, or both) on additivity vs.potentiation of intracellular killing within neutrophils. In parallel,aliquots from each sample will be diluted into sodium polyanetholsulfonate buffer to discontinue peptide-mediated killing, andquantitatively cultured to corroborate flow cytometry analyses ofintracellular killing. Note that peptides, analogues thereof (seeabove), and PMP-2 are compared for relative influences on rabbit andhuman neutrophil intracellular killing of pathogens. Thus, specificdeterminants integral to or selective for potentiation of neutrophilintracellular killing can be identified for further characterization asoutlined below.

If a peptides is found to potentiate neutrophil phagocytosis orintracellular killing of microorganisms, it can be determined whetheroxidative burst is linked to this effect. The generation of reactiveoxygen intermediates such as superoxide anion is considered essential toneutrophil microbicidal potency. Hydroethidine (HE; Molecular Probes)can be used to quantify the influence of peptide on generation ofsuperoxide anion by neutrophils. Neutrophils accumulate HE in thecytoplasm; it is oxidized to ethidium bromide by superoxide anion. Thus,ethidium bromide excitation at 488 nm yields 590 nm emission correlatingwith superoxide anion production, and can be used as detailed below.

Neutrophils isolated as above can be labeled by incubation in RPMIcontaining 1 μM HE for 15 minutes at 37° C. Residual HE is washed away,and neutrophils are exposed to 1-5 μg of selected PMP-2 domains forpredetermined times (0, 15, 30, 60, or 120 mins) at 37° C. in RPME±10%homologous pooled normal serum. The principal variables in theseexperiments are: i) peptides with different structures (e.g., ±C-X-Cmotif); ii) varying durations and concentrations of neutrophil exposureto peptides; and iii) neutrophil priming by peptide followed by exposureto microorganisms. For these experiments, peptides can be selected thatenhance microorganism phagocytosis and/or intracellular killing byneutrophils identified above. Each experiment includes labeledneutrophils in buffer alone (to control for background superoxide anionlevels) in comparison to neutrophils exposed to selected peptides, withor without organisms. Calibration curves based on flow cytometric datafrom known superoxide concentrations using xanthine oxidase assays areused to estimate the absolute superoxide anion levels withinneutrophils. Additionally, selected peptide analogues above are used toascertain the specificity with which they stimulate neutrophil oxidativeburst.

Examples of Novel Antimicrobial Peptides that Act Directly on Pathogensto Exert Microbicidal or Tvhcrobiostatic Activity

Three basic groups can be categorized based on a source and/or designapproach:

-   -   A. Rational Peptides (RP)    -   B. Fragment Peptides (FX)    -   C. Consensus Peptides (CS)

These groups are described in the present application; they are notrecognized categorizations. The majority of peptide sequences listedherein fall into one of these groups.

Examples of Novel Antimicrobial Peptides that Potentiate One or MoreAntimicrobial Acnvmes of Leukocytes

These peptides are derived from domains found in PMPs or other moleculesthat are either known to or predicted to stimulate one or more of theinherent antimicrobial functions of leukocytes such as neutrophils,monocytes, macrophages, and/or lymphocytes. Example sequences in thiscategory are:

PMP-2₁₋₂₂: (SEQ ID NO: 96) Ser Asp Asp Pro Lys Glu Ser Glu Gly Asp LeuHis Cys Val Cys Val Lys Thr Thr Ser Leu Val; PMP-2₁₋₃₇: (SEQ ID NO: 97)Ser Asp Asp Pro Lys Glu Ser Glu Gly Asp LeuHis Cys Val Cys Val Lys Thr Thr Ser Leu ValArg Pro Arg His Ile Thr Asn Leu Glu Leu Ile Lys Ala Gly Gly; andSEQUENCE No. 17 (e.g. RP-15).

Variants of the above sequences or those present in FIG. 12, which havethe described modifications in their Glu-Leu-Arg (ELR) and/or sixthbasic residue components may also be suitable. Examples include:

21-K-PMP-2₁₋₂₂: (SEQ ID NO: 98)Ser Asp Asp Pro Lys Glu Ser Gly Gly Asp Leu His Cys Val Cys ValLys Thr Thr Ser Lys Val; ELR-PMP-2₁₋₂₂: (SEQ ID NO: 99)Ser Asp Pro Lys Glu Ser Glu Gly Glu Leu Arg Cys Val Cys ValLys Thr Thr Ser Leu Val; 21-K, ELR-PMP-2₁₋₂₂: (SEQ ID NO: 100)Ser Asp Asp Pro Lys Glu Ser Glu Gly Glu Leu Arg Cys Val CysVal Lys Thr Thr Ser Lys Val; 21-K, CC-PMP-2₁₋₂₂: (SEQ ID NO: 101)Ser Asp Asp Pro Lys Glu Ser Glu Gly Asp Leu His Cys Cys ValLys Thr Thr Ser Lys Val; ELR, CC-PMP-2₁₋₂₂: (SEQ ID NO: 102)Ser Asp Asp Pro Lys Glu Ser Glu Gly Glu Leu Arg Cys Cys ValLys Thr Thr Ser Leu Val; and 21-K, ELR, CC-PMP-2₁₋₂₂: (SEQ ID NO: 103)Ser Asp Asp Pro Lys Glu Ser Glu Gly Glu Leu Arg Cys CysVal Lys Thr Thr Ser Lys Val.

Further examples include any extension, truncation, substitution,retromerization, fusion, or conformer restriction of these peptides,related templates, or their iterations derived as discussed above. Notethat the full-length PMP-2 is also included in this category bydefinition of its demonstrated inherent leukocyte potentiatingproperties as is illustrated in FIG. 13, showing the chemotactic indexfor rabbit PMP-2 [rPMP-2].

Examples of Novel Antimicrobial Peptide Mosaics that Combine the AboveActivities

These include logical and/or strategic mosaic constructs of the abovepeptides in the categories above. Conceptually, these mosaic peptideswill consist of one or more domains exerting direct microbicidal and/ormicrobiostatic activity linked or otherwise combined with one or moredomains exerting leukocyte potentiating activities. Examples (only a fewof the logical constructs achievable from combining the above peptides)are listed below:

RP-1/PMP2₁₋₂₂: (SEQ ID NO: 104)Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu LeuLys Ser Leu Lys Arg Leu Gly Ser Asp Asp ProLys Glu Ser Glu Gly Asp Leu His Cys Val Cys Val Lys Thr Thr Ser Leu Val;RP-11/PMP2₁₋₂₂: (SEQ ID NO: 105)Ala Leu Tyr Lys Arg Leu Phe Lys Lys Leu LysLys Phe Ser Asp Asp Pro Lys Glu Ser Glu GlyAsp Leu His Cys Val Cys Val Lys Thr Thr Ser Leu Val; andRP-1/21-K, ELR-PMP2₁₋₂₂: (SEQ ID NO: 106)Ala Leu Try Lys Lys Phe Lys Lys Lys Leu LeuLys Ser Leu Lys Arg Leu Gly Ser Asp Asp ProLys Glu Ser Glu Gly Glu Leu Arg Cys Val Cys Val Lys Thr Thr Ser Lys Val.

Other examples of mosaic constructs include any extension, truncation,substitution, retromerization, fusion, or conformer restriction of thesepeptides, related templates, or their iterations derived as outlinedherein.

The antimicrobial peptides and derived metapeptides active alone or incombination with other agents against organisms such as bacteria andfungi can thus comprise peptides having amino acid sequences selectedfrom the group consisting essentially of a first peptide templateXZBZBXBXB and derivatives thereof selected from the group consisting ofXZBBZBXBXB, BXZXB, BXZXZXB, XBBXZXBBX, and BBXZBBXZ, and a secondpeptide template XBBXX and derivatives thereof selected from the groupconsisting of XBBXBBX, XBBXXBBX, BXXBXXB, XBBZXX, XBBZXXBB, andXBBZXXBBXXZBBX. B can be, for example, at least one positively chargedamino acid; X can be, for example, at least one non-polar, hydrophobicamino acid; and Z can be, for example, at least one aromatic amino acid.For example, B can be selected from the group of amino acids consistingof lysine, arginine, histidine, and combinations thereof X can beselected from the group of amino acids consisting of leucine,isoleucine, alanine, valine, and combinations thereof and Z can beselected from the group of amino acids consisting of phenylalanine,tryptophan, tyrosine and combinations thereof. Other amino acids,including glutamine, asparagine, proline, cystine, aspartic acid,glutamic acid, glycine, methionine, serine and threonine, may beinterplaced within these primary structural motifs in a given case.Despite these variations, the disclosed peptides will adhere to thegeneral structural motifs indicated, thereby preserving theiruniqueness.

The first peptide template XZBZBXBXB corresponds to the peptide templateRP-1, Sequence No. 3; and the second peptide template XBBXX correspondsto the peptide template RP-13, Sequence No. 14.

The antimicrobial peptides and derived metapeptides that potentiateantimicrobial activity of leukocytes and are active alone or incombination with other agents directly against organisms such asbacteria and fungi can thus comprise peptides having ammo acid sequencesselected from the group consisting essentially of combined amino acidsequences AL and LA, wherein A represents an antimicrobial domainconsisting essentially of a first peptide template XZBZBXBXB andderivatives thereof selected from the group consisting of XZBBZBXBXB,BXZXB, BXZXZXB, XBBXZXBBX, and BBXZBBXZ, and a second peptide templateXBBXX and derivatives thereof selected from the group consisting ofXBBXBBX, XBBXXBBX, BXXBXXB, XBBZXX, XBBZXXBB, and XBBZXXBBXXZBBX and Lrepresents a leukocyte potentiating domain consisting essentially ofJJJCJCJJJJJJ, and J is selected from X, Z and B. Thus, an example of ALcan be: XZBZBXBXBJJJCJCJJJJJJ; and an example of LA can be:JJJCJCJJJJJJXZBZBXBXB.

The method for developing the novel antimicrobial peptides according tothe principles of the invention is summarized in the flow chart shown inFIG. 3. Initially, the antimicrobial peptide database is inspectedvisually, and the literature is reviewed, utilizing comparative sequencetechniques, in order to identify likely antimicrobial peptide domains.Cidokinins (peptide domains associated with antimicrobial activity) andtoxokinins (peptide domains associated with mammalian cell toxicity) areorganized and domains and structural motifs are identified, and modeledto maximize the cidokinins and minimize the toxokinins. Similarly,immunopotentiating and directly microbicidal peptides may be derived inthis manner. From this modeling, template designs such as RP-1 (SequenceNo. 3), RP-13 (Sequence No. 14), or others are devised, and in turn areused for remodeling, by testing for toxicity, structure andantimicrobial activity, to identify promising candidates for furtherevaluation in vivo.

The antimicrobial peptides of the invention can include truncations,extensions, combinations, mosaics, or fusions of any of the abovetemplate peptides (e.g., PMP-2, Sequence No. 1), analogues derived fromthe approaches contained herein (e.g., RP-1 or Sequence No. 3), ormodified analogues thereof as described above. Examples of suchtruncation, extension, combination, mosaic, and/or fusion sequences aredescribed below:

A. Truncation Example:

PMP-2 (Sequence No. 1) is a 74 residue (amino acids 1-74) antimicrobialpeptide. Novel antimicrobial peptides may be derived from truncation ofPMP-2 (Sequence No. 1), or any of the peptides or their derivativesdescribed herein. For example, the novel effective antimicrobial peptideFX, Sequence No. 30, is a truncation of PMP-2, Sequence No. 1, utilizingresidues 45-74:

Ile Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gin                                    10Ala Ala Leu Tyr Lys Lys Lys Ile Ile Lys Lys Leu Leu Glu Ser                20

Another novel effective antimicrobial peptide resulting from truncationof PMP-2, Sequence No. 1, is PMP-2 residues 28-74 (F28-74, Sequence No.31, with 47 residues; linear/fold; internal fragment) having thefollowing sequence:

Thr Asn Leu Glu Leu Ile Lys Ala Gly Gly His Cys Pro Thr Ala Asn                                    10Leu Ile Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln            20                                      30Ala Ala Leu Tyr Lys Lys Lys Ile Ile Lys Lys Leu Leu Glu Ser                            40

Another novel effective antimicrobial peptide that is a truncationfragment of PMP-2, Sequence No. 1, is PMP-2 residues 43-74 (F43-74,Sequence No. 32, with 32 residues; linear; internal fragment) having thefollowing sequence:

Asn Leu Ile Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu                                    10Gln Ala Ala Leu Tyr Lys Lys Lys Ile Ile Lys Lys Leu Leu Glu Ser            20                                      30

Another novel effective antimicrobial peptide derived by truncation ofPMP-2, Sequence No. 1, is PMP-2 residues 59-74 (F59-74, Sequence No. 33,with 16 residues; linear; internal fragment):

Gln Ala Ala Leu Tyr Lys Lys Lys Ile Ile Lys Lys Leu Leu Glu Ser                                10

B. Extension Example:

RP-1 (Sequence No. 3) is an 18 residue antimicrobial peptide. Novelantimicrobial peptides may be derived from extension of RP-1 or any ofthe other peptides, fragments, or derivatives described herein.

For example, the novel antimicrobial peptide RP-1 extension by RP-1residues 1-10 (RP-1+RP-1-10, Sequence No. 34, having 28 residues;linear; internal fragment) has the following sequence:

Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser Leu Lys Arg                                    10Leu Gly Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu             20

C. Combination/Fusion Example:

RP-1 (Sequence. No. 3) is an 18 residue antimicrobial peptide. RP-13(Sequence No. 14) is a 17 residue antimicrobial peptide. Novelantimicrobial peptides may be derived from combination of RP-1 withRP-13 or any of the other peptides, fragments, or derivatives describedherein.

For example, the novel antimicrobial peptide RP-1 combination with RP-13(RP-1:RP-13, Sequence No. 35, with 35 residues; linear; internalfragment) has the following sequence:

Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser Leu Lys Arg                                    10Leu Gly Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln            20                                      30 Ala Ala Leu

Any of the truncations, extensions, or combinations of any of the abovepeptides may occur in any orientation. For example, an N-terminalportion of RP-1 (Sequence No. 3) may be combined with a C-terminalportion of RP-13. Alternatively, a C-terminal portion of RP-1 may becombined with an N-terminal portion of RP-13. Likewise, other internalfragments may be oriented either N- or C-terminally in any of the abovemodifications.

Further examples of the modifications that can be made to promisingpeptides are set forth below, beginning with various peptides as theparent template to which modifications are made:

RP-1 (parent template, Sequence No. 3):Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser Leu Lys Arg Leu GlyOC-RP-1 (insert Cys at 0, Sequence No. 37):Cys Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser LeuLys Arg Leu Gly 13C-RP-1 (insert Cys at 13, Sequence No. 38):Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Cys Leu Lys Arg Leu Gly19C-RP-1 (insert Cys at 19, Sequence No. 39):Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser Leu Lys Arg Leu Gly CysOC, 19C-RP-1 (insert Cys at 0, 19, Sequence No. 40):Cys Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser Leu Lys Arg Leu Gly CysRP-1-2R (increased + charge, Sequence No. 41):Ala Arg Tyr Lys Lys Phe Lys Lys Lys Leu Leu Lys Ser Leu Lys Arg Leu GlyRP-1-10F (increased steric bulk, Sequence No. 42):Ala Leu Tyr Lys Lys Phe Lys Lys Lys Phe Leu Lys Ser Leu Lys Arg Leu GlyRP-1-2R10F (increased charge, bulk, Sequence No. 43):Ala Arg Tyr Lys Lys Phe Lys Lys Lys Phe Leu Lys Ser Leu Lys Arg Leu GlyRP-1-retro (retromer, Sequence No. 44):Gly Leu Arg Lys Leu Ser Lys Leu Leu Lys Lys Lys Phe Lys Lys Tyr Leu AlaRP-13-retro (retromer, Sequence No. 45):Leu Ala Ala Gln Leu Asp Leu Cys Leu Lys Arg Gly Asn Lys Lys Thr AlanRP-1:cRP-13 (fusion: nRP-1, cRP-13, Sequence No. 46):Ala Leu Tyr Lys Lys Phe Lys Lys Lys Leu Cys Leu Asp Leu Gln Ala Ala LeunRP-13:cRP-1 (fusion: nRP-13, cRP-I, Sequence No. 47):Aia Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Lys Ser Leu Lys Arg Leu Gly

Parent Sequence (1):

PMP-2₄₆₋₆₆ (RP-13-TET, Sequence No. 48):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysLys Charge Conservation Substitution:Lys to Arg (2) (2,3R-RP-13-TET, Sequence No. 49):Ala Thr Arg Arg Asn Gly Arg Arg Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Arg ArgArg Charge Conservation Substitution:Arg to Lys (3) (7K-RP-13-TET, Sequence No. 50):Ala Thr Lys Lys Asn Gly Lys Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysLys Charge Conservation Substitution:Asp to Glu (4) (12E-RP-13-TET, Sequence No. 51):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Glu Leu Gln Ala Ala Leu Tyr Lys LysLys Charge Reversion Substitution:Lys to Glu (5) (3,4,8,19,20,21E-RP-13-TET, Sequence No. 52):Ala Thr Glu Glu Asn Gly Arg Glu Leu Cys Leu Asp Leu Gln Ala Aia Leu Tyr Glu GluGlu Charge Reversion Substitution:Asp to Lys 6) (12K-RP-13-TET, Sequence No. 53):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Lys Leu Gln Ala Ala Leu Tyr Lys LysLys Charge Reversion Substitution:Arg to Glu (7) (7E-RP-13-TET, Sequence No. 54):Ala Thr Lys Lys Asn Gly Glu Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysLys Charge Neutralization Substitution:Arg to Gly (8) (7G-RP-13-TET, Sequence No. 55):Ala Thr Lys Lys Asn Gly Gly Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysLys Charge Neutralization Substitution:Asp to Gly (9) (12G-RP-13-TET, Sequence No. 56):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Gly Leu Gln Ala Ala Leu Tyr Lys LysLys Aromatic Substitution:Tyr to Phe (10) (18F-RP-I3-TET, Sequence No. 57):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Phe Lys LysLys Aromatic Substitution:Tyr to Trp (11) (18W-RP-13-TET, Sequence No. 58):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Trp Lys LysLys Retromer peptide (12) (RP-13-TET-retro, Sequence No. 59):Lys Lys Lys Tyr Leu Ala Ala Gln Leu Asp Leu Cys Leu Lys Arg Gly Asn Lys Lys ThrAla

C-Terminus Truncation:

Parent Seq. (1) (RP-13-TRI, Sequence No. 60):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Conservation Substitution:Lys to Arg (2) (3,4,8,19,20R-RP-13-TRI, Sequence No. 61):Ala Thr Arg Arg Asn Gly Arg Arg Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Arg ArgCharge Conservation Substitution:Arg to Lys (3) (7K-RP-13-TRI, Sequence No. 62):Ala Thr Lys Lys Asn Gly Lys Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Conservation Substitution:Asp to Glu (4) (12E-RP-13-TRI, Sequence No. 63):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Glu Leu Gln Ala Ala Leu Tyr Lys LysCharge Reversion Substitution:Lys to Glu (5) (3,4,8,19,20E-RP-13-TRI, Sequence No. 64):Ala Thr Glu Glu Asn Gly Arg Glu Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Glu GluCharge Reversion Substitution:Asp to Lys (6) (12K-RP-13-TRI, Sequence No. 65):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Lys Leu Gln Ala Ala Leu Tyr Lys LysCharge Reversion Substitution:Arg to Glu (7) (7E-RP-13-TRI, Sequence No. 66):Ala Thr Lys Lys Asn Gly Glu Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Neutralization Substitution:Arg to Gly (8) (7G-RP-13-TRI, Sequence No. 67):Ala Thr Lys Lys Asn Gly Gly Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Neutralization Substitution:Asp to Gly (9) (12G-RP-I3-TRI, Sequence No. 68):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Gly Leu Gln Ala Ala Leu Tyr Lys LysAromatic Substitution: Tyr to Phe (10) (18F-RP-I3-TRI, Sequence No. 69):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Phe Lys LysAromatic Substitution; Tyr to Trp (11) (18W-RP-13-TRI, Sequence No. 70):Ala Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Trp Lys LysRetromer peptide (12) (RP-13-TRI-retro, Sequence No. 71):Lys Lys Lys Tyr Leu Ala Ala Gln Leu Asp Leu Cys Leu Lys Arg Gly Asn Lys Lys Thr

N-Terminus Truncation

RP-50 (Parent Seq. 1, Sequence No. 72):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys Lys LysCharge Conservation Substitution:Lys to Arg (2) (RP-51, Sequence No. 73):Thr Arg Arg Asn Gly Arg Arg Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Arg Arg ArgCharge Conservation Substitution:Arg to Lys (3) (RP-52, Sequence No. 74):Thr Lys Lys Asn Gly Lys Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys Lys LysCharge Conservation Substitution:Asp to Glu (4) (RP-53, Sequence No. 75):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Glu Leu Gln Ala Ala Leu Tyr Lys Lys LysCharge Reversion Substitution: Lys to Glu (5) (RP-54, Sequence No. 76):Thr Glu Glu Asn Gly Arg Glu Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Glu Glu GluCharge Reversion Substitution: Asp to Lys (6) (RP-55, Sequence No. 77):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Lys Leu Gln Ala Ala Leu Tyr Lys Lys LysCharge Reversion Substitution: Arg to Glu (7) (RP-56, Sequence No. 78):Thr Lys Lys Asn Gly Glu Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys Lys LysCharge Neutralization Substitution:Arg to Gly (8) (RP-57, Sequence No. 79):Thr Lys Lys Asn Gly Gly Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys Lys LysCharge Neutralization Substitution:Asp to Gly (9) (RP-58, Sequence No. 80):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Gly Leu Gln Ala Ala Leu Tyr Lys Lys LysAromatic Substitution: Tyr to Phe(10) (RP-59, Sequence No. 81):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Phe Lys Lys LysAromatic Substitution: Tyr to Trp (11) (RP-60, Sequence No. 82):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Trp Lys Lys LysRetromer peptide (12) (RP-61, Sequence No. 83):Lys Lys Tyr Leu Ala Ala Gln Leu Asp Leu Cys Leu Lys Arg Gly Asn Lys Lys Thr Ala

Simultaneous Truncation from Both Directions:

Parent Seq. (1) (RP-62, Sequence No. 84):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Conservation Substitution:Lys to Arg (2) (RP-63, Sequence No. 85):Thr Arg Arg Asn Gly Arg Arg Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Arg ArgCharge Conservation Substitution:Arg to Lys (3) (RP-64, Sequence No. 86):Thr Lys Lys Asn Gly Lys Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Conservation Substitution:Asp to Glu (4) (RP-65, Sequence No. 87):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Glu Leu Gln Aia Ala Leu Tyr Lys LysCharge Reversion Substitution: Lys to Glu (5) (RP-66, Sequence No. 88):Thr Glu Glu Asn Gly Arg Glu Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Glu GluCharge Reversion Substitution: Asp to Lys (6) (RP-67, Sequence No. 89):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Lys Leu Gln Ala Ala Leu Tyr Lys LysCharge Reversion Substitution: Arg to Glu (7) (RP-68, Sequence No. 90):Thr Lys Lys Asn Gly Glu Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Neutralization Substitution:Arg to Gly (8) (RP-69, Sequence No. 91):Thr Lys Lys Asn Gly Gly Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Tyr Lys LysCharge Neutralization Substitution:Asp to Gly (9) (RP-70, Sequence No. 92):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Gly Leu Gln Ala Ala Leu Tyr Lys LysAromatic Substitution: Tyr to Phe (10) (RP-71, Sequence No. 93):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Phe Lys LysAromatic Substitution: Tyr to Trp (11) (RP-72, Sequence No. 94):Thr Lys Lys Asn Gly Arg Lys Leu Cys Leu Asp Leu Gln Ala Ala Leu Trp Lys LysRetromer peptide (12) (RP-73, Sequence No. 95):Lys Lys Tyr Leu Ala Ala Gln Leu Asp Leu Cys Leu Lys Arg Gly Asn Lys Lys Thr

The antimicrobial peptides of the invention can be utilized as 1)individual antimicrobial agents, 2) antimicrobial agents in combinationwith other antimicrobial peptides herein, 3) agents that enhance,potentiate, or restore efficacy of conventional antimicrobials, such asfluoroquinolones, tetracyclines, macrolides, beta-lactams,aminoglycosides, anti-metabolites, azoles, polyenes, or anti-virals, 4)agents that enhance the antimicrobial functions of leukocytes such asneutrophils, 5) prophylactic agents for the prevention of infectiousdiseases, 6) antimicrobial components of vascular catheters orindwelling prosthetic devices, 7) disinfectants or preservatives for usein foods, cosmetics, contact lens solutions, and the like, and 8) agentsto improve efficiency of molecular biology techniques (e.g.,transformation). The novel antimicrobial peptides of the invention can,for example, be formulated in a pharmaceutically acceptable carrier, toform I) powdered or liquid formulations in buffers suitable forintravenous administration, 2) solid or liquid formulations for oraladministration, 3) opthalmalogic solutions or ointments, 4) topicalsolutions or ointments, 5) aerosolized suspensions, lavage, orinhalation formulation, and 6) any combination of the above with medicalinstrumentation or materials. As an example, the mean activity ofseveral peptides according to the invention, in various pharmaceuticallyacceptable carrier solutions, against Staphylococcus aureus andSalmonella typhimurium, is illustrated in FIGS. 4 to 11.

D. Determination of Antimicrobial Peptide In Vitro Activity by Using anAgarose Radial Diffusion Assay Introduction:

The following assay is designed to measure the relative antimicrobialactivity of peptides by determining zones of growth inhibition.

Methods: Antimicrobial Peptide Preparation:

Stock concentrations of antimicrobial peptides are prepared at 1 mg/mLin 0.01% acetic acid are adjusted to pH 7.2.

Media Preparation:

Molecular grade agarose (1.0%) in 10 mM NaH₂PO₄H₂O is autoclaved for 15minutes at 121° C., then held in a waterbath set at 48° C. until use.Mueller Hinton II overlay agarose is prepared by adding molecular gradeagarose to Mueller Hinton II Broth at a final concentration of 1.0%,autoclaving for 10 minutes at 121° C., then holding at 48° C. until use.

Inoculum Preparation:

Trypticase Soy Broth (TSB) (10 mL) is inoculated with overnight growthof the test organism and incubated three to six hours until organismreaches log phase. The cells are collected by centrifugation, washed inPBS, then 0.01% acetic acid adjusted to pH 7.2. The pellet isresuspended in TSB and standardized to a 0.5 McFarland turbiditystandard. A 10 μl aliquot of the inoculum is added to 10 mL of 1.0%molecular grade agarose cooled to 48° C. resulting in a final inoculumconcentration of 5×10⁵ CFU/mL. The suspension is poured into a 15×100 mmpetri dish and allowed to solidify.

After solidification has occurred, five 4 mm diameter wells are boredinto the agarose. The central well is used as the acetic acid controlwhile 10 μA of peptide stock solution is added to each of the other wellresulting in a final concentration of 10 μg peptide/well. The plates areincubated upright for three hours at 37° C., then overlaid with 10 mL ofMueller Hinton II agarose. After the overlay solidifies, the plates areinverted and incubated overnight at 37° C.

Activity Determination:

Zones of growth inhibition are measured. The larger the zone size, thegreater the antimicrobial activity of the peptide. The lack of a zone isan indication of no antimicrobial activity against the target organism.

E. Investigation of the Acute Toxicity of Antimicrobial Peptides in aMurine Model When Administered by a Single Intravenous, Intraperitoneal.Intramuscular or Subcutaneous Injection

Introduction:

The acute toxicity of the antimicrobial peptides can be determined bydosing mice by intravenous (IV), intraperitoneal (IP.), intramuscular(IM.) or subcutaneous (SC.) injection. The highest dose for which theanimals show no signs is considered to be the maximum tolerable does(MTD).

Methods: Test Article Administration:

Swiss CD1 ICRBR male mice of approximately 5-6 weeks of age are weighedand randomized into groups of four mice. The antimicrobial peptide testarticle is administered as a single IV, SC., IM or IP injection to thefirst mouse in each group then the animal is observed for 10 to 30 min.Based on the mortality and morbidity outcome of this administration, thetest article dose, dose volume and route of administration is reassessedbefore the test article is administrated to the next animal. Theindividual dose volume for administration will fall within the range of5-15 mL/kg with the actual dose administered based on the weight of eachanimal on the day of the experiment.

Observations Upon Administration

Each mouse is to be observed 0 to 30 min post administration and againat 1-2, 4-6 and 24 hours. Surviving mice are observed once daily for thenext 6 days. Observations include the activity level of the mouse aswell as any physical side effects of the dose. The maximum tolerabledose (MTD) in mg/Kg is the concentration of peptide for which noobservable adverse effect in noted. Antimicrobial peptides with MTDvalues of >40 mg/Kg are preferred.

It will be apparent from the foregoing that while particular forms ofthe invention have been illustrated and described, various modificationscan be made without departing from the spirit and scope of theinvention.

1-17. (canceled)
 18. An antimicrobial peptide composition for directactivity or for potentiating antimicrobial agents active againstorganisms such as bacteria and fungi, comprising: a peptide of from 13to 74 containing amino acid core sequence selected from the groupconsisting of truncations of PMP-1 (Sequence No. 2), and retromers,extensions, combinations and fusions thereof; truncations of PMP-2(Sequence No. 1), and retromers, extensions, combinations and fusionsthereof.
 19. The antimicrobial peptide composition of claim 18, furthercomprising a pharmaceutically acceptable carrier.
 20. The antimicrobialpeptide composition of claim 18, wherein said peptide is a truncation ofPMP-2 (Sequence No. 1) and comprises residues 28 to 74 of PMP-2(Sequence No. 1).
 21. The antimicrobial peptide composition of claim 18,wherein said peptide is a truncation of PMP-2 (Sequence No. 1) andcomprises residues 43 to 74 of PMP-2 (Sequence No. 1).
 22. Theantimicrobial peptide composition of claim 18, wherein said peptide is atruncation of PMP-2 (Sequence No. 1) and comprises residues 59 to 74 ofPMP-2 (Sequence No. 1).
 23. The antimicrobial peptide composition ofclaim 18, wherein said peptide is a truncation of PMP-2 (Sequence No. 1)and comprises residues 45 to 74 of PMP-2 (Sequence No. 1).
 24. Theantimicrobial peptide composition of claim 18, wherein said peptidecomprises an extension of RP-1 (Sequence No. 3) by RP-1 residues 1-10.25. The antimicrobial peptide composition of claim 18, wherein saidpeptide comprises a combination RP-1 (Sequence No. 3) with RP-13(Sequence No. 14).
 26. An antimicrobial peptide composition for useagainst organisms such as bacteria and fungi, comprising a peptide offrom 8 to 20 amino acids containing an amino acid core sequence of afirst amino acid sequence domain, a second amino acid sequence domain,and a third amino acid sequence domain, where said first amino acidsequence domain is a sequence of from one to six amino acids selectedfrom the group consisting of leucine, isoleucine, alanine, valine,serine, glycine, and threonine; said second amino acid sequence domainis a sequence of from one to two amino acids selected from the groupconsisting of lysine, arginine, histidine, glutamine, proline, glutamicacid, aspartic acid and glycine; said third amino acid sequence domainis a sequence of from one to nine amino acids selected from the groupconsisting of leucine, isoleucine, alanine, valine, serine, glycine, andthreonine: and where the amino acids within said first, second and,third amino acid sequence domains may be separated, and said first,second and third amino acid domains may be separated from each other byup to three amino acids selected from the group consisting ofasparagine, cystine, aspartic acid, glutamic acid and methionine; andretromers, truncations, extensions, combinations, fusions, andderivatives thereof, said peptide having antimicrobial activity.
 27. Theantimicrobial peptide composition of claim 26, wherein said peptidecontains an amino acid core sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇,wherein aa₁ is the amino-terminus of the peptide and is selected fromthe group consisting of leucine, isoleucine, alanine, valine, serine,glycine, and threonine; aa₂ is selected from the group consisting ofleucine, isoleucine, alanine, valine, serine, glycine, and threonine;aa₃ and aa₄ are selected from the group consisting of lysine, arginine,histidine, glutamine, and proline; aa₅ is selected from the groupconsisting of asparagine, cystine, aspartic acid, glutamic acid andmethionine; aa₆ is selected from the group consisting of leucine,isoleucine, alanine, valine, serine, glycine, and threonine; aa₇ isselected from the group consisting of lysine; arginine, histidine,glutamine, proline, alutamic acid, aspartic acid and glycine; aa₅ isselected from the group consisting of lysine, arginine, histidine,glutamine, proline and glutamic acid; aa₉, aa₁₁, aa₁₃, aa₁₅, aa₁₆, andaa₁₇ are selected from the group consisting of leucine, isoleucine,alanine, valine, serine, glycine, and threonine; aa₁₀ and aa₁₂ areselected from the group consisting of asparagine, cystine, asparticacid, glutamic acid and methionine; and aa₁₄ is selected from the groupconsisting of lysine, arginine, histidine, glutamine and proline. 28.The antimicrobial peptide composition of claim 26, wherein said peptidecontains an amino acid core sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇,wherein aa₁ is the amino-terminus of the peptide core sequence and isalanine; aa₂ is threonine: aa₃ and aa₄ are lysine; aa₅ is asparagine;aa₆ is glycine; aa₇ is arginine; aa₈ is lysine; aa₉, aa₁₁, aa₁₃, andaa₁₇ are leucine; aa₁₀ is cystine; aa₁₂ is aspartic acid; aa₁₄ isglutamine and aa₁₅ and aa₁₆, are alanine.
 29. The antimicrobial peptidecomposition of claim 26, wherein said peptide contains an amino acidcore sequence aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈, wherein aa₁ is theamino-terminus of the peptide core sequence and is arginine; aa₂ isphenylalanine; aa₃ is glutamic acid; aa₄ is lysine; aa₅ is serine; aa₆is lysine; aa₇ is isoleucine; and aa₈ is lysine.
 30. The antimicrobialpeptide composition of claim 26, wherein said peptide contains an aminoacid core sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀,wherein aa₁ is the amino-terminus of the peptide and is serine; aa₂ isalanine; aa₃ is isoleucine; aa₄ is histidine; aa₅ is proline; aa₆ andaa₇ are serine; aa₈ is isoleucine; aa₉ is leucine; aa₁₀ is lysine; aa₁₁is leucine; aa₁₂ is glutamic acid; aa₁₃ is valine; aa₁₄ is isoleucine;aa₁₅ is cystine; aa₁₆ is isoleucine; aa₁₇ is glycine; aa₁₈ is valine;aa₁₉ is leucine; and aa₂₀ is glutamine.
 31. The antimicrobial peptidecomposition of claim 26, wherein said peptide contains an amino acidcore sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄, whereinaa₁ is the amino-terminus of the peptide and is tyrosine; aa₂ isalanine; aa₃ is selected from the group consisting of aspartic acid andglutamic acid; aa₄ and aa₅ are selected from the group consisting ofleucine, arginine and histidine; aa₆ is cystine; aa₇ is selected fromthe group consisting of threonine or valine; aa₈ is cystine; aa₉ isserine; aa₁₀ is isoleucine; aa₁₁ is lysine; aa₁₂ is alanine; aa₁₃ isglutamic acid; and aa₁₄ is valine.
 32. An antimicrobial peptidecomposition for use against organisms such as bacteria and fungi,comprising: a peptide of from 5 to 150 amino acids containing an aminoacid core sequence of a first amino acid sequence domain, a second aminoacid sequence domain, a third amino acid sequence domain, and a fourthamino acid sequence domain, and wherein said first amino acid sequencedomain is at the amino-terminus of the amino acid core sequence and is asequence of from one to five amino acids selected from the groupconsisting of phenylalanine, tryptophan, tyrosine, where amino acids ofsaid first amino acid sequence domain may be separated from each otherby an amino acid selected from the group consisting of leucine,isoleucine, alanine; valine and serine; said second amino acid sequencedomain is an amino acid selected from the group consisting of lysine,arginine, histidine, glutamine, and proline; said third amino acidsequence domain is a sequence of from one to five amino acids selectedfrom the group consisting of phenylalanine, tryptophan, tyrosine; andsaid fourth amino acid sequence domain is an amino acid selected fromthe group consisting of lysine, arginine, histidine, glutamine, andproline; and retromers, truncations, extensions, combinations, fusions,and derivatives thereof, said peptide having antimicrobial activity. 33.The antimicrobial peptide composition of claim 32, wherein said peptidecontains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁ is theamino-terminus of the peptide and is lysine; aa₂ is phenylalanine; aa₃is lysine; aa₄ is histidine; aa₅ is tyrosine; aa₆ and aa₇ arephenylalanine; aa₈ is tryptophan; aa₉ is lysine; aa₁₀ is tyrosine; andaa₁₁ is lysine.
 34. The antimicrobial peptide composition of claim 32,wherein said peptide contains the amino acid sequenceaa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁ is theamino-terminus of the peptide and is lysine; aa₂ is glycine; aa₃ istyrosine; aa₄ is phenylalanine; aa₅ is tyrosine; aa₆ is phenylalanine;aa₇ is leucine; aa₈ is phenylalanine; aa₉ is lysine; aa₁₀ isphenylalanine; and aa₁₁ is lysine.
 35. The antimicrobial peptidecomposition of claim 32, wherein said peptide contains the amino acidsequence aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁, wherein aa₁ isthe amino-terminus of the peptide and is lysine; aa₂ is tryptophan; aa₃is lysine; aa₄, aa₅, aa₆, aa₇, and aa₈ are tryptophan; aa₉ is lysine;aa₁₀ is tryptophan; and aa₁₁ is lysine. 36-66. (canceled)